我们要感谢Jennifer Bair和Einat Snir以及爱荷华大学人类遗传学研究所协助准备和处理样品。

所有程序均获得西北大学机构动物护理与使用委员会（IACUC）的批准。 1.电生理解决方案的准备（4小时） <ol><li>通过向999mL反渗透纯化的H 2 O中加入1mL焦碳酸二乙酯（DEPC），使0.1％DEPC处理的H 2 O充分混合，使混合物在室温（RT）下孵育1小时。然后，将DEPC混合的H 2 O在液体循环下高压灭菌15分钟。让DEPC处理的H 2 O在室温下冷却。 </li><li>通过将一瓶Ames培养基和1.9g（23mM）碳酸氢钠混合到1L H 2 O中来制备细胞外溶液。将细胞外溶液与 95％O 2 /5％CO 2 ，并保持在7.3-7.4的pH。 </li><li>通过将125mM的葡萄糖酸钾，2mM MgCl 2，10mM EGTA，10mM HEPES和0.1％DEPC处理的H2 O.将其在-20℃下保存在1mL等分试样中。在每个实验开始时加入10μM的荧光示踪剂。 </li><li>通过向4.15mL细胞外溶液中加入10,000单位胶原酶和83mg透明质酸酶制成酶溶液。酶溶液应在-20°C下保存在50μL等分试样中。 </li></ol> 2.制备视网膜组织（2 h） 注意:本节中的所有步骤都应在昏暗的红色照明下进行<ol><li>在解剖前至少1小时将动物进行黑暗适应。在昏暗的红色照明下执行所有程序。 </li><li>通过CO 2窒息将动物安乐死，并将眼球摘除成具有先前含氧的细胞外溶液的培养皿。 </li><li>用针刺角膜，并用眼科剪刀在角膜和巩膜边缘切割切开。 </li><li>取下镜头＃5镊子。用镊子轻轻敲打巩膜，切断视网膜和巩膜视网膜。小心地从视网膜上清除巩膜。 </li><li>使用＃5镊子去除透明玻璃体;一旦去除，玻璃体就像凝胶状物质粘在镊子上。切片视网膜一半（以便有4件/动物），并将其储存在含氧细胞外溶液中，直至使用。 </li><li> 准备将组织装入记录室时，放置一片视网膜，在500μL含氧细胞外溶液中稀释的酶溶液中孵育。在培养皿中用振荡器在室温下孵育2分钟。 <ol><li>在含氧细胞外溶液中洗涤视网膜，并将组织放置在玻璃底部记录室中;使用塑料转移移液管切断切口以允许视网膜转移而不会损坏组织。 </li></ol></li><li>用于哔哔哔哔哔哔哔咕咕咕。。。。。。。。。。使用移液器去除多余的液体。使用带有尼龙网的铂金环将组织固定。 注意:该方法也可用于制备用于从标记的无远端和双极细胞分离RNA的组织。 </li><li>向室内注入含氧细胞外溶液，并将其安装在显微镜载物台上。用2-4mL / min的氧合细胞外溶液灌注组织。 </li></ol> GFP +视网膜神经节细胞的可视化和靶向（10分钟） 注意:本节中的所有步骤都应在昏暗的红色照明下进行<ol><li>在开始之前，使用微量移液管拉拔器拉动玻璃微量移液器（OD:1.2 mm，ID:0.69 mm）进行电生理记录。对于电极使用以下协议（请注意，参数应相应调整以达到所需的电阻和要求拉杆和玻璃不同）:热量:斜坡+10;拉:0; Vel:23;延迟:1;压力:500;程序循环:5次。确保尖端直径约为1μm，针对大电池的电阻为2-4MΩ，针对较小电池的电阻为5-7MΩ。 </li><li>使用红外差分干涉对比（IR-DIC）光学观察神经节细胞层（ 图1A ）。使用落射荧光（〜480nm）鉴定GFP +视网膜神经节细胞（RGC）（ 图1B ）。 </li><li>找到在DIC中充满细胞内溶液的移液管。在放大器上施加轻微的正压和零电压偏移。 </li><li>将玻璃微量移液管压靠GFP +细胞并施加负压，以在移液管和细胞膜之间形成GΩ密封。应用测试电压指令步骤（ 例如 5 mV）来监测密封电阻。形成稳定的密封后，通过施加n的短脉冲使膜破裂获得全细胞接入的特殊压力。 </li><li>等待1-2分钟，细胞的树突填充荧光示踪剂。 注意:可以通过检查落射荧光中的形态来形成细胞（ 图1C ）。在表达黑素素的RGCs的情况下，通过在荧光照射下检查充满荧光示踪剂的树突并确定它们是否远离离子腺体（M1 ipRGCs），神经节附近的肌瘤分层，来显现内丛状层中的树突状分层（M2＆M4 ipRGCs）或两者（M3 ipRGCs）中的细胞层。这一观察结果与soma大小（M4s与所有其他ipRGC亚型相比具有明显的大体积），允许识别细胞类型20,21,22 。因此，该技术允许在 RNA异构体之前在体外鉴定细胞类型特征研。对于涉及树突形态或细胞生理学的其他细胞类型鉴定方案，可以修饰该方法。 </li></ol>细胞分离（2分钟） <ol><li>开始之前，将台式微量离心机设置为2,000 x g。通过连接管（OD:3/32 in，ID:1/32 in）与1cc注射器制备样品排出装置。 </li><li>将含有10μL裂解缓冲液和1％β-巯基乙醇的0.2mL PCR管置于冰上。准备一个含有DEPC处理的H 2 O的1cc注射器，以冲洗移液器吸头。准备一个干冰容器，以收集样品后冻结裂解缓冲液。 </li><li> 通过使用10mL注射器施加负压，小心地提取细胞移液管的细胞质含量;如果可能，应提取所有细胞质含量，包括细胞器。 <ol><li>通过可视化细胞体尺寸减小来监测DIC中的提取。提取后<html>细胞质内容物，将移液器小心地从组织上提起，并迅速从溶液中移出移液管。 </li></ol></li><li>使用1 mL注射器，用DEPC处理过的H 2 O，从头架上快速移除移液器，然后冲洗移液器末端。通过紧密的管道将移液器连接到1 mL注射器以排出样品。 </li><li> 立即将细胞排出到在0.2mL PCR管中的含有1％β-巯基乙醇的10μL裂解缓冲液1中。 注意:细胞的全部吸出物应轻轻排出，以免引起气泡。 <ol><li>在2,000 xg的桌面微型离心机中将管子简单地离心10秒。立即在干冰上将样品快速冷冻5分钟。冷冻后，将其储存在-80°C最长达两周，以获得最佳效果;样品可能会持续更长时间，但建议尽可能快地进行处理。 </li></ol></li></ol>e。&gt; 5。RNA纯化（30分钟） <ol><li>在开始之前，通过将倒置的P20或P200尖端保持器的顶部贴在96孔磁性支架23上来设置磁选机。 </li><li>准备新鲜的70％乙醇（EtOH） - 每个样品约1 mL即可。从4°C存储中取出RNA磁珠，并在室温下解冻至少30分钟。 注意:一次应该处理不超过8个样品，本协议中的多个步骤依赖于效率和快速处理。 </li><li>一旦磁珠在室温下，旋转30秒，以确保溶液充分混合。 注意:珠使用RNA特异性缓冲液选择性结合RNA，并且当使用磁性支架时，它们允许除去其他细胞废物。 </li><li>在室温下解冻细胞1分钟，然后向每个样品中加入5μL无RNA酶的H 2 O;移液器上下移动。向每个管和移液管中加入22μLRNA珠e彻底混合在室温下孵育样品5分钟，使RNA与磁珠相互作用并结合。 </li><li>将管放在磁选机上放置8分钟;在进行前，确保上清液清澈。从一个颗粒观察珠粒，并确保在移液时不要将其分离。 </li><li>从样品中取出上清液，加入150μL70％EtOH。取出乙醇并重复洗涤两次。 </li><li>让样品风干6分钟。间歇检查，看看是否有更多的乙醇已经收集在管的底部。相应地删除它。 </li><li>当样品干燥时，通过将19μL裂解缓冲液2和1μLRNA酶抑制剂（40U /μL）组合，制备10X反应缓冲液。简单地旋转下来，保持在冰上。 </li><li>一旦样品干燥，珠粒不再具有光泽，从磁选机中取出管，并加入9.5μL无RNase的H 2 O以再次水化样品。将样品放在冰上，并加入1μL10x反应缓冲液至每个样品。 </li></ol>反转录（10分钟） 注意:开始之前，在冰上解冻所需的逆转录试剂（RT，除了酶）。这些包括:引物II，缓冲液1，寡核苷酸和核糖核酸酶抑制剂。 <ol><li>加入2μL引物II（AAGCAGTGGTATCAACGCAGAGTACT （30） N - N，其中N -1可以是A，C或G，N可以是A，C，G或T;12μM）。将管放置在已经在72℃预热3分钟的热循环仪中。 </li><li>孵育期间，准备RT主混合物。对于每个反应，加入4μL缓冲液1（250mM Tris-HCl，pH 8.3; 375mM KCl;和30mM MgCl 2 ），1μL寡核苷酸（48μM）和0.5μLRNA酶抑制剂（40U / μL）。 </li><li>孵化后立即放置管子冰2分钟</li><li>每次反相转录酶（100 U /μL）每次反应加入主混合液中，并彻底移液。向每个管中加入7.5μL主混合物，轻轻移液混匀。简单旋转管子收集底部的内容物，并将它们放入预热的热循环仪中，其程序如下:42℃90分钟，70℃10分钟，保持在4℃。 </li><li>在继续操作之前，将管保存在-20°CO / N，尽管建议在长时间储存​​之前将样品放入放大步骤;其他来源表明在4°C的O / N储存也将在本步24获得接受。 </li></ol> 7. cDNA扩增（2.5h） 注意:开始之前，将PCR缓冲液和PCR引物在冰上融化，然后将其放入台式微型离心机中，然后再进行PCR主混合。 <ol><li>对于每个反应，p重复PCR反应混合物，其中含有25μLPCR缓冲液，1μLPCR引物（12μM），1μLDNA聚合酶和3μL无核酸酶的H 2 O.加入DNA聚合酶，最后加入的主混合样品。 </li><li>向每个管中加入30μL的主混合物，并以2,000 x g的速度旋转10秒，以收集管底部的物质。 </li><li>将管放置在预热的热循环仪中，程序如下:95℃1分钟; 34个循环，98℃10秒，65℃30秒，68℃3分钟; 72℃10分钟;并保持在4°C。 注意:该PCR的循环次数已增加到34，与建议的制造商的说明不同。经过重复测试，发现这个循环次数一直是可靠的结果。 </li><li>将管子保存在-20°C长达一年，然后继续。 </li></ol> 8.扩增cDN的纯化A（30分钟） <ol><li>开始纯化之前，将DNA珠和洗脱缓冲液置于室温至少30分钟。准备新鲜的80％乙醇;每个样品1 mL就足够了。向每个样品中加入1μL10X裂解缓冲液。 </li><li>涡旋DNA珠30秒，并添加50μLDNA珠每个样品。通过移液彻底混合，然后短暂地将它们以2,000xg旋转10秒。在室温下孵育管8分钟。 </li><li>将管置于磁选机上5分钟。将上清液上下移动两次，使样品静置2分钟。当样品在磁性设备上时，取出上清液并丢弃。 </li><li>向每个样品中加入新鲜制备的80％EtOH150μL，使其在室温下静置30秒。取出乙醇并重复EtOH洗涤一次。 </li><li>简单旋转样品并将其放回磁选机1分钟。取出任何剩余的EtOH，并让样品风干5分钟。 </li><li&gt;间歇检查样品，查看是否已经收集了EtOH，并用移液器将其取出。 </li><li>一旦珠粒不再有光泽，但在裂纹开始出现之前，从磁选机中取出管，并加入17μL洗脱缓冲液。轻轻移液上下重新悬挂珠子。 </li><li>将再悬浮样品在室温下孵育2分钟。 </li><li>简单旋转样品以收集底部的所有液体并将其放在磁选机上2分钟。将15μL澄清的上清液转移到1.5 mL无RNA酶的管中，并将其储存在-20°C。 </li><li>使用1μLcDNA检测具有高灵敏度生物分析仪芯片的cDNA文库的质量和大小。 cDNA文库的理想尺寸为0.3-2K​​b，浓度至少为10ng /μL（ 图2 ）。 注意:您可以选择使用PCR作为筛选样品的方法，以确保在进行样品前进行已知标记的表达eparation。 </li></ol> 9.确定浓度和标记cDNA（20分钟） <ol><li>开始之前，将测定试剂，稀释缓冲液和标准品置于室温下30分钟。每次反应准备含有1μL测定试剂和199μL稀释缓冲液的主混合物。将等分试样将199μL该混合物加入到500μL测定管中。加入1μL样品并彻底混匀。 </li><li>将190μL主混合物等分至管1和2。将10μL标准品1加入管1和10μL标准品2加入管2中。将所有样品管和标准管旋转5秒;在室温下孵育3分钟。 </li><li>用高灵敏度荧光计测定样品的浓度。通过选择"计算库存解决方案"选项并突出显示"1μL"，确保已输入正确的样品量。将每个样品在不含RNase的H 2 O中稀释到单独的1.5 mL管中，最终浓度为0.2 ng /μL。 注意:而制造商的指示表明，应该以1 ng /μL的DNA总量开始，我们使用较小的起始量，并且还调整了该修订的议定书。 </li><li>在新的0.2 mL PCR管中，移液2.5μL缓冲液2.将1.25μLcDNA加入合适的管中，总共250 pg。最后，加入每个管中加入1.25μL的标签混合液，移液管彻底混匀;标记混合物内的转座酶将将DNA分成短链，并将衔接子退火至每条链的任一末端，供随后由测序仪使用。 注意:用于标签和索引耦合的卷是制造商说明建议的量的一小部分。这不仅可以保护试剂，而且还可以在我们的经验中一直生产标签样品。 </li><li>将对照离心机上的管子离心1分钟。 </li><li>放管在具有以下程序的预热式热循环仪中:55℃10分钟并保持在10℃。 注意:该孵育的时间为10分钟，而不是从制造商的说明书提出的5分钟。 </li><li>程序完成后立即取出试管，每个加入1.25μL标记中和缓冲液。移液好，混合并在室温下孵育5分钟。立即完成此步骤，因为转座酶保持活性，直到缓冲液中和酶并停止反应。 </li></ol>指数偶联和纯化（1小时） 注意:开始之前，将DNA珠和再悬浮缓冲液置于室温下至少30分钟。决定每个样品使用哪些指标。 注意:这些指数将附加到碎片DNA的相应5&#39;和3&#39;端，以便在测序后鉴定样品。确保没有两个对于可能一起排序的样品，配对是相同的。例如，如果样本1将使用指数白色1和橙色1，样本2应使用白色1和橙色2或白色2和橙色2，但从不相同的索引组合。该套件包含4个不同的白色和6个不同的橙色指数。所有不同的可能组合允许在一个测序通道中集合多达24个样品。虽然我们通常只在泳道中集合10个样品，但也可以使用包含24个指数的试剂盒，如果需要，可以在单个测序通道中汇集96个样品。 <ol><li>对于每个管，添加1.25μL左侧索引和1.25μL正确的索引为该特定样品。加入3.75μLPCR主混合物和移液管，以进行混合。 </li><li>在台面微量离心机中离心1分钟。 </li><li>将管放置在预热的热循环仪中，程序如下:72℃3分钟; 95℃30秒; 95个循环76℃，10秒钟，50℃30秒，72℃1分钟; 72℃5分钟;并保持在4°C。 注意:第95°C的步骤从98°C改变，这是制造商的指示。此外，调整循环细节，使得允许我们样品的最佳标记的温度和时间长度。最后的72℃孵育也被添加到我们的方案中，并不包括在制造商的说明书中。 </li><li>简单旋转管子收集底部的内容物。涡旋DNA珠30秒，然后向每个管中加入30μL，并通过移液彻底混合。 </li><li>在室温下孵育样品5分钟。 </li><li>将样品放在磁选机上2分钟。将上清液上下移动两次，孵育样品1分钟。 </li><li>取出并丢弃澄清的上清液。向每个样品中加入150μL的80％EtOH并除去。重复EtOH洗涤一次。 </li><li>允许the样品空气干燥10分钟。间歇检查以查看是否有任何乙醇已经收集在管的底部，并根据需要去除。 </li><li>一旦裂纹开始出现在DNA珠粒中，则从磁选机中取出样品，加入27.5μL的再悬浮缓冲液以使沉淀物再水化。确保沉淀完全重悬，然后在室温下孵育2分钟。 注意:用于再悬浮缓冲液的体积进行了修改，以解释我们协议中起始材料的数量较少，并且与制造商说明书中的建议体积不同。 </li><li>将管放在磁选机上2分钟。将25μL澄清的上清液转移到无RNA酶的管中，并在-20°C储存。 注意:不要继续遵循制造商的说明来完成库规范化。在该步骤后成功制备样品，并按原样进行测序。 </li><li>分析t使用生物分析仪对样品进行破坏，每个样品1μL。 注意:涂抹分析将会像以前一样进行;然而，现在应该在0.2-1Kb的尺寸范围内检测cDNA（ 图3 ）。低于此点的涂片可能代表样品的降解，而较大的涂片表明不完整的标签。 </li></ol> 11.样品汇集（10分钟） <ol><li>用早期的高灵敏度荧光计获得标记样品的浓度，并以nM计确定浓度。 注意:该计算可以使用互联网转换工具计算，并且依赖于生物分析仪跟踪的平均片段长度。为了确定平均片段长度，分别观察每个样品的痕量，并确定平均DNA片段的大小。通过突出显示涂片的范围并检查生物分析仪的痕迹，也可以计算出这一点e程序计算的摩尔浓度。 </li><li>组合样品使得池中每个样品含有相同的浓度。不要稀释样品;该池应仅与最低浓度样品一样稀释，理想地具有至少15nM的总浓度。 </li><li>测序前将储存的样品储存在-20°C;建议样品在汇集后1周内进行测序。使用HiSeq平台进行配对末端，100 bp测序。 </li></ol>

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M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cone+pathway+influence+on+intrinsically+photosensitive+retinal+ganglion+cell+subtypes.">Differential cone pathway influence on intrinsically photosensitive retinal ganglion cell subtypes.</a> J Neurosci. 30 (48), 16262-16271 (2010).</li><li>Clontech Laboratories I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SMARTerTM Ultra Low RNA Kit for Illumina&#174; Sequencing. , (2013).</li><li>Trombetta, J. J., Gennert, D., Lu, D., Satija, R., Shalek, A. K., Regev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+single-cell+RNA-Seq+libraries+for+next+generation+sequencing.">Preparation of single-cell RNA-Seq libraries for next generation sequencing.</a> Curr. Protoc. Mol. Biol. 4 (22), 1-17 (2014).</li><li>Ecker, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanopsin-expressing+retinal+ganglion-cell+photoreceptors:+Cellular+diversity+and+role+in+pattern+vision.">Melanopsin-expressing retinal ganglion-cell photoreceptors: Cellular diversity and role in pattern vision.</a> Neuron. 67 (1), 49-60 (2010).</li><li>Schmidt, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Role+for+Melanopsin+in+Alpha+Retinal+Ganglion+Cells+and+Contrast+Detection.">A Role for Melanopsin in Alpha Retinal Ganglion Cells and Contrast Detection.</a> Neuron. 82 (4), 781-788 (2014).</li><li>Sanes, J. R., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Types+of+Retinal+Ganglion+Cells:+Current+Status+and+Implications+for+Neuronal+Classification.">The Types of Retinal Ganglion Cells: Current Status and Implications for Neuronal Classification.</a> Annu. Rev. Neurosci. 38, 221-246 (2014).</li><li>Goetz, J. J., Trimarchi, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+Profiling+of+Developing+and+Mature+Retinal+Neurons.">Single-cell Profiling of Developing and Mature Retinal Neurons.</a> J. Vis. Exp. , e3824 (2012).</li><li>Trimarchi, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Heterogeneity+of+Developing+Retinal+Ganglion+and+Amacrine+Cells+Revealed+through+Single+Cell+Gene+Expression+Profiling.">Molecular Heterogeneity of Developing Retinal Ganglion and Amacrine Cells Revealed through Single Cell Gene Expression Profiling.</a> J. Comp. Neurol. 502, 1047-1065 (2007).</li></ol>

作者没有什么可以披露的。

我们的协议通过一个快速易用的指南，证明了一种方法，用于准备用于高质量测序的识别形态学类别的单个细胞，对样品几乎没有损伤。在本手稿中，本征感光性视网膜神经节细胞形态学表征，分离并制备用于RNA-Seq。细胞应激可能发生在视网膜处理过程中;因此，在使用不超过4小时之后，我们更换每块纸巾。我们可以通过使用电生理学装置从这些细胞记录并监测其反应来评估细胞的状态，这使得我们能够确保细胞的健康状况良好。我们的方案允许从23个样品中的15个生成成功的cDNA文库，成功率为65％。此外，我们的测序数据质量很好，因为我们的细胞表达的基因的平均数目为2,316-10353，平均为5,683基因注册非零FPKM值。这些数字与由神经元5的其他研究发现的表达的基因计数类似，表明我们的数据质量也很好。 虽然我们已经通过这个协议为这些特定的神经元取得了成功，但是应该注意的是，这种技术的微小修改可以应用于许多不同的应用。在单独的小鼠模型中使用荧光激活的细胞分选器（FACS），我们分离了来自中枢神经系统的GFP标记物标记的小量细胞（1,000-225,000）。分离来自这些种群的RNA，并且在步骤6.1中使用1μL（在或略低于12ng /μL）的该RNA开始逆转录。在步骤7.4之后，应该对协议进行唯一的调整，此时可以选择使用更少的PCR周期。当初始样品含有g时，我们已经使用了19个循环重新发现超过10,000个细胞，并且发现这产生了优质的cDNA文库。例如，细胞样品是从FACSorted种群制备的，所表达的基因数目范围为12,340-14,052，平均为13,265个具有非零FPKM值的基因。该技术可以应用于存在荧光报告物的各种小鼠模型。由于这一修正案，研究人员可能会研究任何一种荧光报告小鼠可用的细胞群，从而延伸了神经元多样性领域的研究。 可以基于功能而不仅仅是形态来对图形单元进行第二调整。该项目依赖于转基因模型，但是这种技术可以应用于没有已知分子识别符的神经元。例如，目前鉴定的视网膜神经节细胞有30多种功能亚型，其中很少有独特的标记。作为这种技术已经依靠用于细胞填充的电生理学装置，可以使用补片夹紧来基于其尖峰图案来识别每个细胞的功能反应谱。使用光诱发的穗记录，可以在细胞分离之前确定视网膜神经元的分类。这种技术适用于视网膜神经节细胞，但可以扩展到检查其他视网膜神经元。然而，应当注意的是，如果该方案用于在内核层中形成视网膜双极或无长突细胞，例如，必须小心在电极的尖端处提供恒定的正压，以避免接触细胞作为电极通过神经节细胞层和内丛状层。对于内核层内的视网膜神经元，在细胞记录之前的视网膜切片的制备可能最有效地始终避免污染。每个单元将按照如下所述的分离和制备制版步骤此外，我们提出使用该方案进行神经元的研究，但是该技术可以应用于通过使用转基因模型可形态表征或分离的任何细胞类型。 这种技术也可以被修改为与以前制备的cDNA文库一起使用，就像我们过去在微阵列杂交中所产生的一样。使用单独制备的文库，一个开始于50-150ng范围内的cDNA量，并在步骤8.4开始方案;没有尝试更高和更低的浓度，尽管它们也可以工作。首先使用DNA珠纯化cDNA，然后进行标记和PCR扩增。我们用来自文库制备物的cDNA（如微阵列杂交29）测试了这种调整，并且已经成功地产生了用于RNA-Seq的标记文库。 鳍该协议可用于评估来自诱导的多能干细胞（iPSC）的特定诱导细胞群的成功。这些重新编程的多能细胞的分化背后的驱动力依赖于导致细胞类型彼此分开开发的转录因子的理解。将iPSCs推向特定的细胞命运是研究神经元多样性的新方法，因为它可用于选择性地产生细胞类型。该方案可以适应于从该模型中评估分化细胞之间的多样性质量。如前所述，视网膜神经节细胞有30多种功能亚型，并且已经做出许多努力来产生来自iPSC的功能性RGC。鉴定RGC亚型的标记 - 在我们的例子中，ipRGCs - 将允许研究人员评估其方案在生产各种RGCs亚型方面的成功。细胞类型的评估这些神经元中的这些神经元将受益于该方案，并且还可以作为继续研究亚型特异性标记的工具，因为该模型系统变得容易获得。 在本手稿中，我们描述了用于分离和制备用于转录组学分析的单细胞的简单技术。此外，我们建议编辑协议的方法，以便将此技术用于考虑到一系列目标的大量实验。这里描述的协议由Trombetta 等人描述的方案改编24作为低输入RNA-Seq的推荐试剂盒说明书的调整。主要的区别在于细胞隔离和各种体积变化，我们选择最适合本项目的需要。重要的是要强调，本议定书中最重要的步骤是将细胞从视网膜组织中分离出来。这是在durin协议中的要点g哪些大多数错误都可以发生，应该非常仔细地注意。任何数量的污染可能导致不可用的样品，而细胞质的损失可能导致mRNA分子的严重消耗。该步骤应小心进行，并由同一个人从实验到实验进行，以确保样品处理精确。 总之，该协议描述了用于高质量RNA-Seq的细胞的分类，分离和制备的技术。这是一种高效，相对低成本的方法来优化单个单元的数据质量。这种技术是多功能的，并且可以被最小程度地修改用于各种研究。

在整个中枢神经系统中观察到神经元多样性，特别是在脊椎动物视网膜中，这是一种由1个神经胶质细胞和6个神经元细胞类型组成的高度专门化的组织，其由视网膜祖细胞1,2,3组产生 。细胞的许多亚型可以在功能上，形态上和遗传上分类。该方案的目标是将细胞类型的遗传变异性与其可识别的功能和/或形态特征相结合。已经鉴定了许多基因用于细胞分类，但是许多亚型仍然没有表征，因为它们代表总体群体的一小部分。这些特定亚型中的基因的鉴定将允许更好地了解视网膜内的神经元多样性，并且还可以揭示其他地方的神经细胞的多样化。福此外，单细胞研究允许揭示新细胞类型，这可能被忽略，因为它们在总体4,5,6,7中的代表性很低。 单细胞转录组学的优点之一是可以发现定义特定细胞亚型的独特标记或标记组合。然后，这些可用于获得对该细胞类型的遗传接触以进行不同的操作。例如，我们正在使用该协议来表征视网膜神经节细胞亚类的细胞类型特异性基因，其表达色素黑素素。荧光标记物在表达黑色素的视网膜神经节细胞中的使用使得能够研究这些细胞，因为它们由于其已知基因的表达而聚集在一起。有趣的是，这种细胞群有五种已知的亚型老鼠视网膜中的信号8 。因此，为了从每种类型的细胞中分离RNA，我们在转基因模型中已经使用确定的形态学分类，以在细胞分离之前鉴定每种亚型。这种技术允许表征细胞以及直接从视网膜分离，而不需要组织解离，这可能导致细胞内的应激反应和由于切断的树突引起的污染9 。 随着RNA-Seq方法的不断发展，过去几年中已经有了许多新技术。这些工具允许最大化的细胞获取和更高的成本效率，同时接近手头4，7，10，11，12，13的问题。但是，虽然这些技术一直是优秀的踏脚石，还有一些障碍仍然遇到这个协议能够解决。首先，许多目前的程序将细胞与解离的组织分离，并尝试使用主成分分析或事后分层聚类来确定细胞分类。依靠这些工具来分类亚型可能不会产生可靠的结果，并且可能迫使人们找到新的方法来验证这一数据，以便将遗传标记与功能性细胞类型相关联。其他方案中解离的要求有时会导致组织损伤，并可能导致神经元过程被切断，导致mRNA潜在的丧失。此外，在解离的细胞制剂中，应激反应可能开始影响这些细胞的转录组14 。该协议通过确定隔离前的功能细胞类型来克服这些挑战，并且更好地维持h通过保持视网膜组织完整而保持细胞。 2014年引入了一种技术，其中包括活细胞转录组的体内分析。虽然这种技术允许以对组织的最小的机械破坏来检查转录组，但是在检查其转录组之前没有使用非常特异性的记录小鼠，其缺乏对组织内的特定细胞类型进行分类的能力。我们的方案不需要特定的记者，因为我们利用细胞填充和电生理学在细胞分离前表征细胞。此前协议的另一个限制是需要特定的波长来激发可光活化的元件，而我们的方案允许使用荧光报告物和荧光染料，这些荧光染料可以容易地获得，也可以由每个实验室单独选择。然而，其他实验室却结婚了两种电话生物学和转录组学研究细胞多样性。已经在分离的神经元16上进行使用膜片钳记录来表征细胞在其分离之前的功能，并且在某些情况下，已经使用微阵列分析17进行这些研究。这些方法遇到相同的并发症，因为它们需要组织解离或使用微阵列技术，其依赖于样品与可用探针的杂交。最近的进展之一是Patch-Seq的发展，Patch-Seq是一种结合使用膜片钳记录和RNA-Seq技术来了解全脑切片细胞的技术。虽然这种技术与本文提出的方案有相似之处，但重要的是要注意，我们的方法允许组织保持完整，以保持细胞的健康。在这里，我们提出了一个优化协议该联盟产生高质量的单细胞文库以使用RNA-Seq获得高读取深度和测绘覆盖率。

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Ames&#39; Medium</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:108px;">A1420-10X1L</td>
			<td style="width:372px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-gluconate</td>
			<td>Spectrum Chemical</td>
			<td>PO178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 594 Hydrazide</td>
			<td>Invitrogen</td>
			<td>A10442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS005273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyaluronidase</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS002592</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish (35mm diameter)</td>
			<td>Thermo Fisher Scientific</td>
			<td>153066</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmologic scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">#5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Shaker</td>
			<td>Fisher Scientific</td>
			<td>13-687-708</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Micropipette</td>
			<td>Sutter</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micropipette Puller</td>
			<td>Sutter</td>
			<td></td>
			<td>P-1000 horizontal pipette puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1mL syringe</td>
			<td>Fisher Scientific</td>
			<td>14-823-2F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flexible tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCL lysis buffer</td>
			<td>Qiagen</td>
			<td>1031576</td>
			<td>Lysis Buffer 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free Water</td>
			<td>Qiagen</td>
			<td>129112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml PCR tubes</td>
			<td>Eppendorf</td>
			<td>30124359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl Alcohol, Pure</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td>Ethanol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analog Vortex Mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>02215365</td>
			<td>Vortex</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt RNAClean XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63987</td>
			<td>RNA magnetic beads</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MagnaBlot II Magnetic Separator</td>
			<td>Promega</td>
			<td>V8351</td>
			<td>Magnetic stand</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml MCT Graduated Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Smart-Seq v4 Ultra Low Input RNA Kit</td>
			<td>Clontech</td>
			<td>634888</td>
			<td>Reagents for Reverse Transcription and PCR Amplification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Lysis Buffer</td>
			<td></td>
			<td></td>
			<td>Lysis Buffer 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5X Ultra Low First-Strand Buffer</td>
			<td></td>
			<td></td>
			<td>Buffer 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3&#39; SMART-Seq CDS Primer II A</td>
			<td></td>
			<td></td>
			<td>Primer II</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SMART-Seq v4 Oligonucleotide</td>
			<td></td>
			<td></td>
			<td>Oligonucleotide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SMARTScribe Rverse Transcriptase</td>
			<td></td>
			<td></td>
			<td>Reverse Transcriptase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2X SeqAmp PCR Buffer</td>
			<td></td>
			<td></td>
			<td>PCR Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Primer II A</td>
			<td></td>
			<td></td>
			<td>PCR Primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SeqAmp DNA Polymerase</td>
			<td></td>
			<td></td>
			<td>DNA Polymerase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mastercycler pro S</td>
			<td>Eppendorf</td>
			<td>950030020</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>DNA magnetic beads</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HS Bioanalyzer Chips &#38; Reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32851</td>
			<td>For the calculation of sample concentrations</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit Assay Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit 2.0 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT DNA Sample Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>Reagents for Tagmentation and Index Coupling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TD Buffer</td>
			<td></td>
			<td></td>
			<td>Buffer 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATM</td>
			<td></td>
			<td></td>
			<td>Tagmentation Mix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NT Buffer</td>
			<td></td>
			<td></td>
			<td>Tagmentation Neutralizing Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NPM</td>
			<td></td>
			<td></td>
			<td>PCR Master Mix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td>Indices for Tagmentation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N501</td>
			<td></td>
			<td></td>
			<td>White 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N502</td>
			<td></td>
			<td></td>
			<td>White 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N701</td>
			<td></td>
			<td></td>
			<td>Orange 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N702</td>
			<td></td>
			<td></td>
			<td>Orange 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiSeq 2500</td>
			<td>Illumina</td>
			<td>SY-401-2501</td>
			<td>For completing sequencing of samples</td>
		</tr>
	</tbody>
</table>

single-cell rna-seq of defined subsets of retinal ganglion cells

细胞类型特异性标记的发现可以提供对细胞功能的了解和细胞异质性的起源。随着对神经元多样性的更好理解的推动，识别表达定义各种细胞亚群的基因是重要的。视网膜作为中枢神经系统多样性研究的优秀模式，由多种主要细胞类型组成。每个主要类别细胞的研究已经产生了便于识别这些群体的遗传标记。然而，在这些主要视网膜细胞类别的每一个中存在多种细胞亚型，并且这些亚型中很少有已知的遗传标记，尽管许多已经通过形态或功能表征。对于个别视网膜亚型的遗传标记的知识将允许与特定视觉功能相关的脑靶标的研究和绘图，并且还可以洞察基因网络维持细胞多样性。用于鉴定亚型遗传标记的现有途径具有缺点，例如测序后细胞类型的分类。这为数据分析提出了挑战，需要严格的验证方法来确保集群包含相同功能的单元格。我们提出了一种用于在分离和测序之前鉴定细胞的形态和功能的技术，这将允许更容易鉴定亚型特异性标记。这种技术可以扩展到非神经元细胞类型，以及稀有的具有微小变异的细胞群体。该协议产生优质的数据，因为许多库为单个单元提供了大于2000万次读取的读取深度。该方法克服了单细胞RNA-Seq提出的许多障碍，并且可能适用于以直接和高效的方式分析细胞类型的研究人员。

细胞类型在染料注射后容易分类 图1显示荧光示踪剂填充之前和之后的GFP + RGC的实例。基于其在转基因品系中GFP的表达鉴定该细胞（ 图1A ）。在细胞的细胞上形成细小的拉丝玻璃电极的密封。为了表征亚型，将荧光染料注入到血浆中并允许填充所有相关的过程（ 图1B ）。通过观察，该细胞具有非常大的血细胞瘤，并且其过程终止于内丛状层的ON亚细胞膜（ 图1C ）中，可以使用M4 ipRGC进行分类。作为在分离的视网膜制剂中可以看出的分层差异的例证，我们填充了M1（OFF-stratifying），M3（双组分）和具有荧光示踪剂的M4（ON分层）ipRGC，固定它们，并进行ON和OFF亚花层的共聚焦成像（ 图1D ）。通过共聚焦成像的树突的这种可视化与当细胞填充荧光示踪剂时树突在未固定的体外组织中的看法非常相似（Schmidt和Kofuji 2009,2010，2011）。 来自单细胞的cDNA文库使用Oligo d（T）引物可以进行mRNA的选择性逆转录和扩增。在生成和扩增每个细胞的cDNA文库后，使用生物分析仪芯片来评估文库的质量和大小。该分析的结果表明，在良好的样品中理想的cDNA涂片为0.5-2Kb（ 图2A ）。一些样品在300 bp标记之下显示几个条带或涂片怀疑这些库的质量差（ 参见表1进行故障排除）。优质生物分析仪痕迹的一个例子显示出低于300bp的DNA条带或500bp和2Kb之间的鲁棒涂片（图2B）。空控制通道应分别显示35和10380 bp的较低和较高标记，以及不会波动的稳定基线。各种图书馆可以产生质量数据， 如图2B和2C 所示 ，其产生了优异的读取深度和高映射率。只有具有预期尺寸的强cDNA文库的样品才能进行标记（ 参见表1进行故障排除）。 低输入标签准备高质量样品进行测序该协议针对具有少量起始材料的标签进行了优化<html>湖只有250 pg最适合成功的标签，因为较低的量不会正确放大，更高的数量将不会完全碎片化。完成标记和PCR扩增/样品清除后，再次在生物分析仪芯片上分析样品。此时理想的cDNA涂片应为0.2-1Kb（ 图3A ）。痕迹应该平滑地均匀分布在这两种尺寸之间（ 图3B ）;该轨迹对应于车道1中的样本。 图3C显示了不完整的标签，可以轻松解决（ 参见表1进行故障排除）。我们对样品进行了数据分析，发现该方案生产的样品具有优异的读取深度，通常每个样品超过1200万个读数;平均为69.1％的绘图;超过5000个表达基因。 <html> /55229fig1.jpg"/&gt; 图1:基于表达黑曲霉的ipRGCs中GFP表达的RGC类型的鉴定和形态特征。 （ A ）视网膜的神经节细胞层，在整体视网膜制剂中使用IR-DIC可视化。 （ B ） 相同的制备物在表型荧光（〜480nm）中显示以鉴定GFP表达的神经节细胞。 （ C ）用于膜片钳记录并填充荧光示踪剂的GFP表达细胞。基于其非常大的体积大小和分层的树突25,26 ，可以将细胞鉴定为M4 ipRGC。 （ D ）在IPL（M4）的ON子衬底中以及在IPL（M3）的ON和OFF子层中成像在IPL（M1）的ON和/或OFF子衬底中的ipRGC树突的共聚焦图像。 IR-DIC:红外差分干涉对比度。.jove.com / files / ftp_upload / 55229 / 55229fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" src="/files/ftp_upload/55229/55229fig2.jpg" /> 图2:用生物分析仪分析cDNA文库质量。 （ A）已经反向转录，扩增和纯化的多个样品的生物分析仪输出实例。泳道1-3显示理想的DNA涂片，大多数DNA大于300bp。具有此范围涂片的图像库一直提供优异的测序数据，平均每个细胞表达5,683个基因。泳道4代表未成功处理的样品，因此不产生cDNA。成功的对照泳道在35和10380bp具有恒定的基线和两个清洁的峰。 （ B ）在2Kb左右的高强度cDNA的成功生物分析仪追踪的实例。这是<html> mear对应于泳道1中的样品。（ C ）成功的生物分析仪跟踪的示例，其中以500bp为中心的cDNA。该轨迹对应于车道3中的样本。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55229/55229fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55229/55229fig3.jpg" /> 图3:标记样品显示0.2和2 Kb之间的坚固涂片。 （ A ）标签，扩增和PCR清除后的代表性实例生物分析仪输出。 （ B ）对应于（A）中的泳道1的成功标记样品的踪迹。 （ C ）具有不完整标签的样品的迹线实例，以1Kb左右的峰强度清除。"&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>问题</td><td>可能的原因</td><td colspan="2">解</td></tr><tr><td>步骤3.3）不能形成GΩ密封</td><td>电池表面不够干净</td><td colspan="2">使用正压进一步清洁</td></tr><tr><td>步骤3.3）细胞似乎放气/死亡</td><td></td><td colspan="2">准备新的移液器并定位一个新的细胞</td></tr><tr><td>步骤3.4）无法确定树突的末端位置</td><td>阿列福克没有足够的时间来扩散整个细胞或浓缩的Alexafluor不够高</td><td colspan="2">检查以确保相机上的增益和曝光时间足够高。再等待5分钟，然后再观察树突。如果树突仍然不可见，请用较高的Alexafluor制备溶液浓度。 </td></tr><tr><td>步骤4.1）无法吸出所有细胞质</td><td></td><td colspan="2"></td></tr><tr><td>步骤5.5）8分钟后上清不清楚</td><td></td><td colspan="2">轻轻移液整个溶液两次，同时仍然在磁选机上，再放置5分钟</td></tr><tr><td>步骤5.5）吸液期间颗粒分散</td><td>样品离磁铁太远</td><td colspan="2">在所有移液过程中将管保持在磁分离装置上。排出溶液并允许珠粒重新沉淀5分钟</td></tr><tr><td>步骤5.7）5分钟后，样品仍然有光泽</td><td>最大量的EtOH未被除去</td><td colspan="2">在干燥过程中继续监测样品。每2分钟一次，使用P10移液管，并取出管底部的所有EtOH </td></tr><tr><td>步骤8.9）在补液之前，颗粒有裂纹</td><td></td><td colspan="2">允许样品再水化总共4分钟，而不是2（步骤8.10） </td></tr><tr><td>步骤8.11）用上清液培养少量珠粒</td><td></td><td colspan="2">将整个样品放回管中放置在磁选机上1分钟;轻轻取出，确保避免沉淀</td></tr><tr><td>步骤8.12）DNA涂片不一致，荧光标度不断变化</td><td> HS芯片的DNA浓度过高</td><td colspan="2">检查样品浓度，稀释介于1-10ng /μL之间。重新运行生物分析仪</td></tr><tr><td>步骤8.12）低分子量涂片</td><td> RNA降解</td><td colspan="2">确保在收集后立即闪烁冻结细胞。丢弃该cDNA文库</td></tr><tr><td>步骤8.12）对照泳道中波动标记基线</td><td>污染或老试剂</td><td colspan="2">丢弃DNA标记并使用新的生物分析仪管</td></tr><tr><td>步骤8.12）无DNA涂片</td><td>未能驱逐细胞</td><td colspan="2">拉一个稍大一点的新针头</td></tr><tr><td></td><td> RNA珠粒失败</td><td colspan="2">在使用前确保小珠完全重新悬浮，并允许样品在放置在磁选机上之前孵育</td></tr><tr><td>步骤8.12）DNA涂片较弱</td><td>放大不够</td><td colspan="2">采用更多的PCR循环</td></tr><tr><td>步骤8.12）500bp-2Kb范围外的DNA涂片</td><td></td><td colspan="2">低于0.5和2 Kb以下的DNA涂片可能会被污染。舍弃样品</td></tr><tr><td>步骤8.12）DNA迹线上定期间隔的尖峰</td><td>样品污染</td><td colspan="2">一定要穿新鲜的手套，用新的乙醇冲洗;过滤嘴应随时使用</td></tr><tr><td>步骤9.3）无法检测荧光计上样品的浓度</td><html><td></td><td colspan="2">低于检测水平的样品对于标记具有太少的DNA，不能使用</td></tr><tr><td>步骤10.10）10分钟后样品不干燥</td><td>继续除去过量的EtOH </td><td colspan="2">允许样品空气干燥，每分钟检查一次</td></tr><tr><td>步骤10.13）涂抹向2Kb倾斜</td><td>不完整的碎片</td><td colspan="2">重稀释样品浓度为0.15 ng /μL，而不是0.2 ng /μL;重新运行标签</td></tr><tr><td>步骤10.13）涂抹歪向200bp </td><td> DNA输入太少</td><td colspan="2">稀释样品少，尝试浓度为0.4 ng /μL;重新运行标签</td></tr><tr><td></td><td>标签反应太长</td><td colspan="2">将标签反应时间缩短到8分钟</td></tr><tr><td>步骤10.13）薄涂片</td><td>不正确的DNA扩增</td>2"&gt;稀释样品浓度为0.4 ng /μL，并重新运行标签</td></tr><tr><td>步骤11.2）最大可获得的池浓度低于5nM </td><td></td><td colspan="2">确定哪些样品具有显着低的浓度，并用新的稀释液重新运行标记</td></tr></tbody></table> 表1:议定书中潜在障碍的解决方案和建议。此表列出了本协议期间可能发生的潜在困难以及可能遇到的步骤。此表列出了许多这些问题的可能原因，以及可能有助于解决任何问题的解决方案。

Watch this Scientific Journal Video about Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells at JoVE.com

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

在这里，我们提出了一种组合方法，用于在分离之前对神经元细胞类型进行分类，以及随后对单细胞转录组进行表征。该协议优化了用于成功的RNA测序（RNA-Seq）的样品的制备，并描述了专门为增强细胞多样性的理解而设计的方法。

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

single-cell-rna-seq-of-defined-subsets-of-retinal-ganglion-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.7K 次观看.  Iowa State University. 在这里，我们提出了一种组合方法，用于在分离之前对神经元细胞类型进行分类，以及随后对单细胞转录组进行表征。该协议优化了用于成功的RNA测序（RNA-Seq）的样品的制备，并描述了专门为增强细胞多样性的理解而设计的方法。 

视频: Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...