This procedure generates chromatin immunoprecipitation data from dissected fly tissue to accurately reflect chromatin state in vivo drosophila tissue. In this case, hundreds of testes are harvested by hand dissection. Chromatin immunoprecipitation assays are performed using an antibody against the protein of interest.
Follow up quantitative R-T-P-C-R analysis assay for enrichment of the specific genomic region in the purified chipped DNA. Alternatively, high throughput sequencing of the chip sample generates a genome wide profile. Ultimately, chip analysis of dissected tissue provides a representation of the chromatin state in cells from their physiological conditions.
This protocol is optimized for the analysis of tissues directly isolated from the organism. In order for to obtain chromatin states and epigenetic information under real physiological conditions, we'll demonstrate the use of chip in the drosophila in order to look at the crumpton state. Now this protocol can be applied to other organisms such as mouse or zebrafish.
Visual demonstration of this protocol is critical to obtain DNA insufficient quality, quality and quantities in order to use for chip or the generation of sequencing libraries for high throughput sequencing. First, dissect the tissue of interest in this case around 200 pairs of drosophila testes in cold PBS containing protease inhibitor and 100 micrograms per milliliter PMSF. After rinsing the tissue twice, resus suspending 200 microliters of the same PBS solution with inhibitors.
Then to fix the sample at 5.5 microliters of warmed 37%formaldehyde. Incubate at 37 degrees Celsius for 15 minutes. Vortexing every five minutes, allow the tissue to settle on ice.
Rinse twice with 450 microliters of PBS containing inhibitors. Store the samples at minus 20 degrees Celsius. Combine the 200 oph testes samples in a 1.75 milliliter einor tube.
Aspirate the buffer and re suspend the samples. In 200 microliters of lysis buffer. Dissociate the tissue completely with a blue homogenizer, ensuring no aggregates remain incubate room temperature for 10 minutes.
Then fragmentalize the sample with a microchip soat, rest on ice for 50 seconds. Repeat four to five times to obtain optimum fragment size for the chip target. Then dilute with 1.8 milliliters ripper buffer for a stock of chromatin extract For a successful chip assay, it is critical to empirically determine the size of the DNA fragments.
So we need to perform gel electrophoresis for proceeding to the chip experiment. For the input control sample, remove 50 microliters. Add two microliters of five molar sodium chloride and incubate at 65 degrees Celsius overnight.
To reverse cross-link, proceed to conjugate the antibody to the protein. A beads in a 1.5 milliliter fendor tube. Add 40 microliters of protein A beads and 600 microliters of PBS rock at four degrees Celsius for two minutes.
Then apply the beads to the magnet and discard the supinate. Now add 100 microliters of PBS and also the antibody of interest. Incubate it room temperature for one hour.
After harvesting the beads by magnet, add one milliliter of tissue chromatin extract. Rotate four degrees celsius overnight. Next, apply the chip sample to a magnet and remove the supinate.
Proceed to one milliliter washes of harvested beads at four degrees SIUs for 10 minutes each wash, re suspend the beads in 100 microliters of tea buffer. To reverse cross-link the protein DNA complexes, add three microliters of 10%SDS and five microliters of 20 milligrams per milliliter. Proteinase K incubate 65 degrees Celsius overnight.
Pellet the beads on a magnet. Transfer the supinate containing DNA into a new tube. Wash the beads once and combine the 2D NA containing supernatants.
Next, purify the DNA with a phenol chloroform extraction and transfer the upper aqueous layer into a new tube. Precipitate the DNA with 20 micrograms per milliliter, linear acrylamide or glycogen. 20 microliters of three molar sodium acetate and 500 microliters of 100%Ethanol.
Mix well and incubate at minus 80 degrees Celsius for 10 minutes. Pilot the DNA by centrifugation. Discard the supernatant and wash the pellet once with 300 microliters of 70%ethanol after air drying.
Resus, suspend the DNA pellets in 50 microliters of TE buffer. Store the chipped DNA samples at minus 20 degrees Celsius. Prepare serial dilution of genomic DNA for the standard curve.
In a 96 well plate using templates of diluted genomic DNA input control or chipped DNA Set up 20 microliter QPCR in duplicate spin the 96 well plate in the mini plate spinner. Run the sample plate in a realtime PCR system to validate the data. First, verify the presence of a single peak in the thermal dissociation plot indicative of a single amplicon from the PCR.
Next, plot the standard curve. Using data from the series of diluted genomic DNA, then calculate the formula that resolves DNA amount according to the cycle threshold. Ct.Also determine the linear phase of exponential amplification of PCR for each primer set.
If the CT value of the chip DNA and input control samples are within the linear range of CT value, then calculate the enrichment of chip DNA versus input taken into consideration. The dilution factor end repairs 0.1 to five micrograms of chipped DNA in 50 microliter reactions containing T four DNA polymerase and T four polynucleotide kinase. As per manufacturer's instructions incubate for 45 minutes at room temperature.
Then purify the DNA using a MinLu reaction cleanup kit. Continue to add a overhangs to the three prime end with DNA polymerase one large CLA NOW fragment and DATP incubate for 30 minutes at 37 degrees Celsius. Again, pass the reaction over a spin column to purify and concentrate the DNA.
Proceed to ligate with adapter oligo. Mix from Illumina using T four DNA Ligase incubate for one hour of room temperature. Then run the sample through the MinLu reaction cleanup kit.
Eluting the DNA in 20 microliters. Next, separate the DNA sample by electrophoresis using the egel apparatus. Isolate the 200 to 400 base pair band in the gel and purify using the KYOGEN gel extraction kit.
Now amplify a library using paired end primers, fusion HF and pH enzymes from Illumina program 20 PCR cycles. Then on a standard 2%AOSE gel isolate 200 to 400 base pair DNA fragments for high throughput sequencing in Ellen's software. Align all reads to the drosophila genome, allowing up to two mismatches with the reference sequence.
Retain uniquely mapped reads for downstream analysis for multiple identical reads. Retain up to three copies to reduce the possibility of biases from PCR amplification. Next, use a Python script to convert the output of the GA pipeline to browser extensible data files.
Set a four base pair window size and a 160 base pair DNA fragment size. Classify the drosophila genes into silent and express genes by RPKM. Digital number, designate genes with RPKM equal to zero.
As a silent group, classify genes with RPKM greater than one into an expressed group with three subgroups of low, moderate, and high expression levels. Finally, compare the histone modification and gene expression levels using a Python script previously described in basal 2007, the bag of marbles. Mutant testes have failure in the transition from proliferative spermatogonia to differentiating sper cytes.
This chip QPCR experiment uses BAM testes as a source for undifferentiated germ cells to query the chromatin state of key genes required for sperm differentiation, enrichment of histone modification, his stone three lysine 27 methyl three or histone three, lysine four, methyl three. At the differentiation genes is determined by normalization to a constitutively expressed cycling a gene. The differentiation genes male specific transcription factor 87 Don Juan and fuzzy onion, which are not expressed in BAM testes are highly enriched with the repressive histone three, lysine 27 methyl three histone modification.
Furthermore, they're devoid of the active histone. Three, lysine four methyl three histone modification. A chromatin signature we call monovalent in contrast to the valency with both histone three lysine form methyl three and histone three.
Lysine 27 methyl three enriched at differentiation genes in embryonic stem cells. Chip sequence data using the same set of antibodies. Confirm the chip QPCR result shown in figure one A genomic regions of all three tested terminal differentiation genes are highly enriched with three lysine, 27 methyl three, but not with histone three, lysine four methyl three.
In contrast, the constitutively expressed cyclin aging has high histone three lysine, four methyl three, but low histone three lysine 27 methyl three near its transcription start site. This figure shows ChIP-seq data of two histo modifications near the transcription start sites of four differentially expressed gene classes. Histone three, lysine 27 methyl three is enriched downstream of the transcription start sites, which is inversely correlated with gene expression level.
The silent genes exhibit the highest histone three lysine 27 methyl three level highly expressed genes show no histone three lysine 27 methyl three binding consistent with the repressive role of histone three, lysine 27 methyl three on gene expression. Conversely, enrichment of the histone three, lysine four methyl three modification around the transcription start sites showed the opposite correlation with gene expression level consistent with the active role of his stain. Three, lysine four, methyl three on gene expression.
Once mastered, this technique can be performed properly in two to three days. Don't forget to use safety precautions when using phyl, chloroform and formaldehyde. We hope this protocol has given you sufficient understanding of using chromatin immunoprecipitation in order to look at the chromatin landscape in in vivo tissue.
