This manuscript outlines a protocol for preparing an ATAC-seq library of neutrophils from murine bone marrow, aiming to guarantee optimal neutrophil viability and high library quality. It offers step-by-step instructions on BMC preparation, immunomagnetic sorting, and library construction, serving as a valuable guide, especially for newcomers to studying neutrophils.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.

Neutrophils are one of the most abundant and crucial immune cell types in vertebrates, constituting 50%-70% of human leukocytes¹. Neutrophils play a central role in detecting and eliminating microorganisms in hosts. During sepsis, mature neutrophils are among the first responders². They quickly mobilize to infection sites via chemotaxis³, where they combat pathogens by ingesting microorganisms (phagocytosis), producing NADPH oxidase-dependent reactive oxygen species (respiratory bursts), secreting granules (degranulation), and forming neutrophil extracellular traps (NETs) that catch and kill extracellular bacteria once they arrive at the inflammation site⁴. Immature neutrophils are also quickly mobilized from the bone marrow into peripheral circulation by chemokines^5,6. These neutrophils attracted to the infectious site also release cytokines, which are involved in several physiological processes, such as hematopoiesis, angiogenesis, and wound healing^7,8. Patients afflicted with congenital or acquired diseases that result in diminished neutrophil counts are more susceptible to infections^9,10. However, neutrophil activation is a double-edged sword. Excessive activation and cytokine release can cause tissue and organ damage, leading to multiple organ dysfunction if infections are not promptly controlled^11,12. Additionally, neutrophils also contribute to various inflammatory and autoimmune diseases and can influence cancer progression and metastasis^13,14. This underscores the need to study the regulation of neutrophil activity to balance effective immune response and prevent damage to the host.

In neutrophils, the chromatin architecture undergoes distinct and dynamic changes in their differentiation, migration, and activation, exerting pivotal regulatory roles throughout the cellular lifespan. This journey, from the large, round, euchromatin-rich nucleus of myeloblasts to a more indented nucleus in myelocytes and metamyelocytes, and then to a C or S-shaped in-band cells and highly lobulated with densely packed chromatin in polymorphonuclear neutrophils^15,16, is a testament to the complexity of neutrophil biology. The specialized chromatin configuration ensures the selective expression of genes essential for neutrophil function, such as those involved in granule production and rapid transcriptional responses necessary for effective pathogen elimination¹⁷. When neutrophils are mobilized to inflammatory sites via chemotaxis, the transendothelial migration puts pressure on the nucleoskeleton/cytoskeleton complex linker (LINC) complex, prompting chromatin remodeling and more activation potential¹⁸. In sepsis, activated neutrophils exhibit a "nuclear-left shift," with abnormal chromatin morphology, such as bilobed or non-lobed nuclei and significantly looser chromatin condensation¹⁹. This results in heightened accessibility within gene regions associated with inflammatory response, thereby inducing significant expression of these genes. If infections are not duly controlled, histone citrullination and subsequent chromatin deconstruction are necessary to form NETs^20,21. Therefore, understanding neutrophil chromatin architecture is crucial for advancing neutrophil biology and developing treatments for inflammatory and autoimmune diseases, offering hope for future disease treatment.

Chromatin accessibility serves as a metric for chromatin architecture at the epigenomic level. It indicates how genomic regions are physically permissible to enhancers, promoters, insulators, and chromatin-binding factors and how these elements influence gene expression²². Assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a widely used technique to assess chromatin accessibility across the genome²³. It does not require prior knowledge of regulatory elements, making it a potent tool for epigenetic research. This method involves isolating nuclei of samples, fragmenting genomic DNA by transposase Tn5, and adding sequencing primers for library preparation, sequencing, and data analysis²³. ATAC-seq has been instrumental in studying nucleosome mapping, transcription factor binding analysis, regulatory mechanisms in various cell types or diseases, novel enhancer identification, biomarker discovery, etc^22,23. It is often combined with other techniques, such as RNA sequencing, for a comprehensive analysis of gene expression.

ATAC-seq has advanced our knowledge of neutrophil biology by uncovering precise epigenetic mechanisms in health and disease. It has revealed several key transcription factors (i.e., RUNX1, KLF6, PU.1, etc.) in neutrophil maturation and effector responses^24,25, uncovered pathogen-specific signatures with biomarker potential in sepsis²⁶, elucidated the epigenomic mechanisms of the innate immune memory¹², and identified therapeutic targets in pediatric acute myeloid leukemia (AML)²⁷. However, as a prominent cellular component in immune surveillance, neutrophils exhibit high sensitivity to their external milieu and thus pose a challenge to producing high-quality ATAC-seq data. Under physiological circumstances, the median half-life of human neutrophils is reported to be 3.8 days, whereas the average half-life of mouse neutrophils is 12.5 h. It is noteworthy that their half-life is considerably shorter when studied in vitro^28,29. During in vitro operation of isolated neutrophils, exposure to microorganisms or pathogen-associated molecular patterns (PAMPs), as well as redundant experimental procedures and harsh operations, can result in aberrant neutrophil activation and diminished cellular viability due to apoptosis or NETosis. The deceased cells commonly house significant amounts of unchromatinized DNA highly susceptible to Tn5, consequently elevating the background noise of ATAC-seq²³.

Here, we propose a scalable protocol for preparing ATAC-seq libraries optimized for neutrophils derived from rodent bone marrow samples. Figure 1 presents a graphical overview of the protocol, which encompasses the immunomagnetic separation of neutrophils from bone marrow, followed by ATAC-seq. The improved immunomagnetic sorting guarantees the optimal viability of neutrophils before extracting the nucleus for the ATAC-seq library, thus ensuring the high quality of the libraries and facilitating the incorporation of additional neutrophil assessments into the scheme. The implementation of this protocol will assist researchers in understanding the chromatin architecture features and epigenomic reprogramming mechanisms of neutrophils when exposed to various microenvironmental challenges. This is expected to have broad applications in basic and clinical immunology studies.

All animal procedures presented below complied with national ethical and animal welfare guidelines and regulations, which were approved by the Animal Care Research Ethics Committee of the Capital Medical University, Beijing, China (sjtkl11-1x-2022(060)).

1. Preparation of bone marrow cell (BMC) suspensions

1. Select male C57BL/6J mice aged 6-8 weeks weighing 19-21 g. Euthanize the mice using a CO₂ chamber, followed by cervical dislocation after the deep reflex has disappeared. Disinfect them with 70% ethanol spray on a dissection mat.
2. Use small scissors and forceps to carefully separate the femur from the hip joint and tibia, remove the attached skin and skeletal muscle, and rinse the femur with cold phosphate-buffered saline (PBS) in a 10 cm Petri dish.
3. Cut off the femur's epiphyses, gently insert a 28 G needle attached to a 2-mL syringe into the marrow cavity, and flush out the bone marrow with cold PBS containing 2% fetal bovine serum (FBS).
4. Repeat the previous step until the flushed dark red fluid turns translucent. Three flushes are usually required.
   NOTE: Avoid inserting the needle too deeply into the bone marrow cavity to ensure optimal cell acquisition.
5. Use a 70 µm cell strainer to filter the cell suspension into a fluorescence-activated cell sorting (FACS) tube (5 mL).
6. Lyse the erythrocytes in BMCs with a commercial erythrocyte lysis buffer without polyformaldehyde.
   1. Centrifuge the tube at 500 x g for 5 min at 25 °C, carefully remove the supernatant, leaving approximately 100 µL of liquid at the bottom, and gently tap the tube to loosen the pellet.
   2. Add 2 mL of lysis buffer, diluted from erythrocyte lysis buffer (10x), and mix by turning the tube upside down three times. Then, centrifuge the tube at 500 x g for 5 min, discard the supernatant, and resuspend the cell pellet by gentle tapping.
   3. Wash the cells twice with RPMI 1640 medium containing 10% FBS and resuspend them in a 2 mL volume.
      NOTE: When dealing with unscattered cell clumps, it is recommended to use a 200 µL pipette tip to pick them out instead of filtering the cell suspensions through a 70 µm cell strainer. This method can reduce cell loss.
7. Calculate the cellular viability and total number of living cells per sample with trypan blue staining.

2. Immunomagnetic labeling of neutrophils with anti-Ly6G magnetic beads

1. Resuspend the bone marrow cells (~2 x 10⁷ live cells per mouse) in 2 mL of RPMI 1640 medium containing 10% FBS and centrifuge at 500 x g for 5 min at 25 °C.
   NOTE: Given the limited requirement of 1 x 10⁵ cells for ATAC-seq, the surplus neutrophils can be allocated for additional epigenomic analyses (e.g., ChIP-seq and Hi-C) or immune function assessments (e.g., cytokine production, phagocytosis, and NETs) using neutrophils obtained from the same mouse.
2. Aspirate most of the supernatant, leaving behind approximately 100 µL, and gently tap the tube to loosen the pellet.
3. Add 30 µL of Ly6G beads and mix with the BMCs by gentle tapping.
   NOTE: Magnetic beads should be vortexed for a minimum of 10 sec prior to use. Following the addition of the beads to BMCs, it is recommended to refrain from vortexing.
4. Incubate the tube at 25 °C for 15 min.
5. Add 2 mL of PBS to resuspend the beads and cells, then centrifuge at 500 x g for 5 min at 25 °C.
6. Aspirate most of the supernatant, leaving behind approximately 100 µL, and gently tap the tube to loosen the pellet. Add additional PBS for a final volume of 1 mL.
   NOTE: When dealing with unscattered cell clumps, it is recommended to use a 200 µL pipette tip to pick them out instead of filtering the cell suspensions through a 70 µm cell strainer. This method can reduce cell loss.

3. Neutrophil separation with immunomagnetic cell sorting

1. Pre-treat the MS column with PBS.
   1. Position the MS column within the magnetic field of a compatible magnetic-activated cell sorting (MACS) separator. Position an unoccupied 5 mL tube beneath the fluid discharging from the MS column.
   2. Rinse the column with 500 µL of PBS. Repeat this step after the complete flow of PBS into the MS column.
      NOTE: The PBS should be allowed to flow gradually, drop by drop, into the 5 mL fluorescence-activated cell sorting (FACS) tube via the MS column. Ensure that no air enters the column, as air bubbles may obstruct the column.
2. Apply the magnetically labeled BMC suspension to the MS column by adding 500 µL at a time, immediately refilling once the column reservoir is empty.
   NOTE: Ensure that the cell suspensions are devoid of cell mass. If needed, position a new 5 mL FACS tube beneath the MS column to collect the unlabeled cells (e.g., monocytes and lymphocytes) before this stage.
   NOTE: Take note of the droplet flow rate. A slow flow rate could suggest that the sorting column is obstructed.
3. Wash the MS column by adding 500 µL of PBS each time the reservoir is empty, repeating this process twice.
   NOTE: The premature addition of PBS may impede the entry of certain cells into the sorting column, potentially compromising the purity of cell sorting.
4. Collect the magnetically labeled neutrophils.
   1. Once the column reservoir is depleted, remove the column from the magnetic field and carefully position it onto a 5 mL tube.
      NOTE: Prior to column removal, ensure no residual liquid within it to prevent the loss of neutrophils due to droplet discharge during the process. The residue in the column could be removed using a pipette.
   2. Dispense 2 mL of PBS onto the column, then immediately and firmly push the plunger into the column to expel the magnetically labeled neutrophils.
      NOTE: To acquire sufficient neutrophils, ensure adequate elution volume and stay short when pushing the plunger into the column.
5. Use a hemocytometer to enumerate the isolated cells. Identify the purity of the isolated neutrophils by taking 100 µL of the post-sorting cell suspension and analyzing it with flow cytometry.

4. Nucleus extraction

1. Centrifuge the 50,000 single-cell suspensions containing the neutrophils at 500 x g for 5 min at 4 °C in a 0.2 mL tube, then discard the supernatant.
2. Resuspend the neutrophils in 50 µL of pre-cooled Lysis buffer (refer to Table 1), gently mix the solution using a pipette, and incubate on ice for 10 min.
   NOTE: If the sample volume is less than 5 µL, adding the Lysis buffer directly is permissible. Cell suspension concentration does not exceed 1 x 10⁷ cells /mL.
3. Centrifuge at 500 x g for 5 min at 4 °C, then discard the supernatant and collect the nucleus. Position the tube on ice before commencing subsequent procedures.
   NOTE: To minimize sample loss, denote the side of the tube wall where pellets accumulate and gently aspirate the liquid from the opposite side when removing the supernatant.

5. Nucleosome-tethered tagmentation with transposase

1. Prepare the Tagmentation Reaction Mix containing Tn5 transposase in a polymerase chain reaction (PCR) tube according to the guidelines of the DNA Library Prep kit (refer to Table 2) and pre-warm the refrigerated amplicon purification beads at 25 °C for at least 30 min.
2. Resuspend the neutrophil nucleus with 50 µL of tagmentation reaction mix and gently agitate with a pipette, followed by centrifugation for 5s using a handheld centrifuge (~2600 x g).
3. Incubate the reaction tube in a PCR instrument at 37 °C for 30 min.
4. Add 100 µL of pre-vortexed amplicon purification beads to the lysate, then mix the magnetic beads thoroughly in the solution by pipetting up and down 10 times.
   NOTE: Magnetic beads are sticky and can easily attach to the pipette tips, therefore, ensure to aspirate the right volume and slowly dispense them from the tips when transferring the beads.
5. Incubate the reaction tube at 25 °C for 5 min, then centrifuge it for 5 s with a handheld centrifuge (~2600 × g).
6. Purify the interrupted DNA fragment by placing the reaction tube on a magnetic rack until the solution becomes clear (approximately 5 min). Then, carefully remove the supernatant without disturbing the magnetic beads.
7. Wash the magnetic bead with 200 µL of freshly prepared 80% ethanol each time, while keeping the reaction tube on the magnetic rack for 30 s. Avoid disturbing the beads when adding ethanol. Repeat the procedure described in step 5.7.
8. Centrifuge the reaction tube with a handheld centrifuge (approximately 2600 × g), aspirate the ethanol with 10 µL pipette tips, then dry the magnetic beads for 3-5 min while opening the lid.
   NOTE: Drying durations for magnetic beads may vary across different regions due to variations in humidity levels. The beads should be dried just until they lose their shine. Over-drying can complicate the elution process, while inadequate drying may result in the presence of alcohol residue, impacting subsequent experiments. In addition, positioning the magnetic rack horizontally is more conducive to the uniform drying of the magnetic beads.
9. After removing the reaction tube from the magnetic rack, add 26 µL of ddH₂O to cover the magnetic beads, pipetting up and down to ensure thorough mixing. Then, incubate at 25 °C for 2 min.
   NOTE: Extend the incubation duration appropriately if the magnetic beads are over-dried.
10. Briefly centrifuge the reaction tube with a handheld centrifuge (approximately 2600 × g) and separate the magnetic beads using a magnetic rack until the solution becomes clear (approximately 5 min).
11. Carefully transfer the 24 µL supernatant to the new PCR tube.

6. Library construction for next-generation sequencing (NGS)

1. Prepare the PCR Reaction Mix in a sterilized tube utilizing purified DNA fragments and primers containing sequencing adapters (refer to Table 3).
2. Conduct the PCR reaction following the recommended procedures (refer to Table 4), typically requiring approximately 1 h.
   NOTE: The experiment may be temporarily halted after this procedure, and the PCR products could be stored at -20 °C or at least 2 weeks.
3. Add 27.5 µL of pre-vortexed amplicon purification beads to 50 µL of PCR products, then mix the magnetic beads thoroughly in the solution by pipetting up and down 10 times.
   NOTE: The incorrect ratio of magnetic beads to PCR products can result in discrepancies in the lengths of sorted fragments. Use ddH₂O to fill up the evaporated volume of the PCR product to 50 µL. Ensure to aspirate the right volume of magnetic beads and dispense them slowly from the pipette tips to minimize the beads that may be attached.
4. Incubate the reaction tube at 25 °C for 5 min, then centrifuge it for 5 s with a handheld centrifuge (approximately 2600 x g).
5. Purify the library DNA by placing the reaction tube on a magnetic rack until the solution becomes clear (approximately 5 min). Then, carefully transfer the supernatant to a new sterilized PCR tube and discard the magnetic beads.
6. Add 50 µl of pre-vortexed amplicons purification beads to the supernatant, then mix the magnetic beads thoroughly in the solution by pipetting up and down 10 times.
7. Incubate the reaction tube at 25 °C for 5 min, then centrifuge it for 5 s with a handheld centrifuge (approximately 2600 x g).
8. Place the reaction tube on a magnetic rack until the solution becomes clear (approximately 5 min), then carefully remove the supernatant without disturbing the magnetic beads.
9. Wash the magnetic bead twice with 200 µL of freshly prepared 80% ethanol each time, keeping the reaction tube on the magnetic rack for 30 sec. Avoid disturbing the beads when adding ethanol.
10. Dry the magnetic beads for 5 min while opening the lid.
11. After removing the reaction tube from the magnetic rack, add 22 µL of ddH₂O to cover the magnetic beads, pipetting up and down for 10 times to ensure thorough mixing. Then, incubate at 25 °C for 5 min.
12. Briefly centrifuge the reaction tube with a handheld centrifuge (approximately 2600 × g) and separate the magnetic beads using a magnetic rack until the solution becomes clear (approximately 5 min).
13. Carefully transfer the 20 µL supernatant to a new sterilized PCR tube and store it at -20 °C.
    NOTE: To achieve a library with a more concentrated length distribution, consider utilizing a gel recovery kit to sort the amplified product. Alternatively, if there are no specific requirements for the length distribution range, the amplified product can be directly purified using a magnetic bead or column purification kit without length sorting.

7. Library quality control and sequencing

1. Utilize DNA fragment analyzer platforms to assess the length distribution of library DNA.
2. Utilize NGS platforms to perform high-throughput sequencing on the libraries that have passed the quality inspection.

The protocol outlined was employed to isolate neutrophils from the bone marrow of C57BL/6 mice and compare their respective variance in chromatin accessibility pre- and post- lipopolysaccharide (LPS) stimulation via ATAC-seq. Typically, 2 x 10⁷ BMCs can be harvested from both femurs of a 6-8-week-old C57BL/6 mouse, and subsequently, 2-5 x 10⁶ neutrophils can be isolated. Among them, 1 x 10⁵ cells are employed for ATAC-seq, while the surplus cells can be allocated for comprehensive neutrophil analysis.

FACS immunophenotyping was adopted to determine the neutrophil purity and viability after the immunomagnetic sorting (Figure 2). Although the distribution of forward scatter-area (FSC-A) and side scatter-area (SSC-A) alone could provide an estimate, specific neutrophil marker staining was used here to demonstrate the sorting effect. Following the FSC-A and SSC-A gating, cells were further gated using FSC-A and forward scatter height (FSC-H) to distinguish single cells from aggregated ones. Around 99% of post-sorting cells were identified as single. Then, 7-amino-actinomycin D (7AAD), a DNA dye efficiently excreted by live cells, was excluded to label cells and identify >99% of viability from post-sorting single cells. CD11b and CD48 were further labeled to identify myeloid cells and distinguish neutrophils from monocytes within myeloid cells, respectively. In BMCs of C57BL/6 mice, myeloid cells constituted 46.7% of viable cells, with neutrophils accounting for 76.4% of the myeloid cell population. After the immunomagnetic sorting, the neutrophil purity within viable cells was verified to be 98.5%, meeting the requirement of the ATAC-seq library construction.

After constructing the ATAC-seq library, the purified DNA underwent length distribution analysis using the DNA fragment analyzer. The results showed a length between 100 bp and 1000 bp with no small or large fragment contamination (Figure 3A). The concentrations of the ATA-seq libraries and quantities of the sequencing data are detailed in Table 5. After receiving the sequencing data, we also examined the length distribution of ATAC-seq fragments (Figure 3B). Since the tagmentation of Tn5 transposases produces signature size pattern of fragments derived from the nucleosome-free regions (NFR, < 100 bp), mononucleosome (~200 bp), dinucleosome (~400 bp), trinucleosome (~600 bp) and longer oligonucleosome from other open chromatin regions, a successful ATAC-seq experiment should coordinately generate the typical fragment size distribution plot with periodical decreasing peaks corresponding to integer multiples of nucleosomes (Figure 3B). Alterations of ATAC-seq peaks were generally identified to illustrate the dynamic landscape of open chromatin regions. As shown in Figure 3C, the regions with increased or decreased ATAC-seq signals should be depicted by clear heatmaps. We then analyzed the chromatin accessibility of genes for inflammatory factors that are primarily affected by LPS stimulation, such as IL10, Stat1, Il12a, Cxcl1, and Irak1, and we found that these genes had higher accessibility in LPS-stimulated neutrophils (Figure 3D). Taken together, the quality control of the ATAC-seq library we constructed was satisfactory, and the data produced were sufficient to analyze the epigenetic changes of mouse neutrophils after LPS stimulation.

Figure 1: Schematic overview of the protocol for neutrophil ATAC-seq library preparation. The overview follows the preparation of BMCs suspensions, the labeling of neutrophils with anti-Ly6G magnetic beads, the neutrophil separation by immunomagnetic sorting, extraction of the neutrophil nucleus, nucleosome-tethered tagmentation, NGS library construction, and sequencing. Please click here to view a larger version of this figure.

Figure 2: The neutrophil purity and viability assessment using FACS before and after the immunomagnetic sorting. (A) BMCs and (B) isolated neutrophils underwent staining with anti-CD11b, anti-Ly6G, anti-CD48, and 7AAD according to a standardized protocol. Subsequently, single cells were gated from the total cell population, excluding dead cells with 7AAD. Myeloid cells were then gated from live cells, and finally, neutrophils were gated from the myeloid cell population. Please click here to view a larger version of this figure.

Figure 3: Quality assessment of ATAC-seq library prepared from murine bone marrow neutrophils. (A) The length distribution of the library DNA analyzed by the DNA fragment analyzer, and (B) of the ATAC-seq fragment was determined by sequencing data. (C) Pile-up heatmap of genomic regions achieving accessibility (± 10 kb around the center of those peaks) in untreated and LPS-stimulated neutrophils. (D) Integrative genomics viewer (IGV) views of ATAC-seq signals at representative gene loci corresponding to inflammatory factors, where red and black signal peaks represent untreated and LPS-stimulated neutrophils, respectively. Please click here to view a larger version of this figure.

Table 1: Lysis buffer formulation for nucleus extraction.

Table 2: Preparation of Tagmentation Reaction Mix.

Table 3: Preparation of PCR Reaction Mix for NGS library construction.

Table 4: Recommended procedures of PCR reaction for NGS library construction.

Table 5: ATAC-seq Library Concentration and Sequencing Data Overview.

This manuscript reports an experimental protocol for preparing ATAC-seq libraries optimized for neutrophils derived from rodent bone marrow samples. Due to neutrophils being highly susceptible and readily activated immune cells, optimization efforts prioritized maintaining the viability of isolated neutrophils. Improper treatment may lead to NETosis and other forms of cell death, prompting the release of significant quantities of unchromatinized DNA and thereby contributing to the background noise²³. We streamlined the procedure prior to nucleus extraction and chose an erythrocyte lysis buffer without polyformaldehyde. In contrast to peripheral blood samples, a minimal amount of erythrocytes necessitates a brief incubation period of BMCs with the erythrocyte lysis buffer, effectively minimizing the loss of neutrophils while ensuring the thorough dissolution of erythrocytes. To minimize unnecessary stimuli, it is imperative to pre-warm all reagents to 37 °C before incubating with BMCs and to handle them with the utmost care, avoiding activities such as vortexing, repeated filtering with cell strainers, prolonged centrifugation with excessive rotational speed, etc. Enhancements in the procedures above improve the sorted neutrophil viability, thereby creating conditions conducive to high-quality ATAC-seq libraries.

We have explored three widely used sorting techniques to obtain neutrophils with optimal viability: immunomagnetic separation, density gradient centrifugation, and flow cytometry sorting. When conducting density gradient centrifugation, neutrophils can cross the separation fluid layer, potentially leading to osmotic pressure and NET formation. When performing flow cytometry sorting, the neutrophils passing through the nozzle experience stimuli due to mechanical shear force. The sorting duration typically exceeds one hour, and the cellular concentration is diminished post-sorting, resulting in greater neutrophil loss during the separation and subsequent centrifugation. Compared to the two approaches above, immunomagnetic separation achieves a 95% purity and 99.5% viability of neutrophils within 30 min. While this methodology may lead to higher experimental expenses, we consider it essential for acquiring dependable research outcomes. Except for ATAC-seq, immunomagnetic separation can also be integrated with other experiments that have stringent criteria for neutrophil viability, such as immune function assessments (e.g., cytokine production, phagocytosis, NETs, etc.), 'omics' methodologies (e.g., ChIP-seq, Hi-C, broad-spectrum targeted metabolomics, etc.), and other routine molecular biology experiments. The integrated methodology allows for comprehensive studies of neutrophil functions and related mechanisms from various perspectives within a single mouse.

The protocol described is a scalable scheme that can be expanded to accommodate neutrophils from diverse species and various tissue sources. Based on the erythrocyte ratio in the samples, it is advisable to optimize the incubation period with the erythrocyte lysis buffer to the minimum while ensuring sufficient dissolution efficiency. During immunomagnetic sorting, the amount of magnetic beads used significantly impacts the purity of the isolated cells. Optimization should be tailored to the samples' quantity of cells processed and the neutrophil ratio. Maintaining the purity of neutrophils is the primary focus. As per our experience, add 30 µL of beads for 2 x 10⁷ cells from two femurs and 50 µL for 4 x 10⁷ cells from two femurs and two tibias. Due to the excellent reproducibility across different batches and the broad range of antibody dosage, optimization only needs to be explored initially. Additionally, the generation of high-quality and stable ATAC-seq libraries is contingent upon the appropriate ratio of Tn5 transposase to the cellular nucleus invested³⁰. The optimal amount to invest is 50,000 nuclei for murine bone marrow-derived neutrophils, with minor adjustments needed for other samples.

Taken together, we propose an integrated scheme of the immunomagnetic sorting of neutrophils and following ATAC-seq library preparation, which holds great promise for extensive utilization in the investigation of neutrophils. It will aid researchers in comprehending neutrophils' chromatin architecture features, uncovering their regulatory mechanisms of inflammatory response and epigenomic reprogramming in the presence of various microenvironmental challenges, thereby revealing potential therapeutic targets in disease occurrence and progression^{12,24,25,26,27}.

The authors have nothing to disclose.

This work was supported by grants from the R&D Program of the Beijing Municipal Education Commission (KZ202010025041) and the Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY29).