ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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.

Results

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 neutrop...

Discussion

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

Materials

Name	Company	Catalog Number	Comments
0.4% Trypan Blue	Scientific	T10282
1x phosphate-buffered saline	Biosharp	BL302A
20 bp–5000 bp Alignment marker	Bioptic	C109102-100A	DNA fragment analyzer supporting products
5k Size marker(Standard 50 bp Ladder)	Bioptic	C109101-100A	DNA fragment analyzer supporting products
Amplicons Purification Beads	Beckman	A63881	Amplicons Purification Beads is a nucleic acid purification kit for obtaining high quality DNA products.
Anti-Ly6G MicroBeads Ultrapure, mouse	miltenyi	130-120-337	The Anti-Ly-6G MicroBeads UltraPure, mouse were developed for positive selection or depletion of mouse neutrophils from single-cell suspensions of mouse bone marrow.
Anti-mouse CD11b-BV605 (1:200)	BD	Cat#563015
Anti-mouse CD48-PE-Cy7 (1:400)	BD	Cat#560731
Anti-mouse Ly6G-BV510 (1:200)	BD	Cat#740157
C57BL/6J mice, wild type, 8-week-old, male	The Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences	The Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences
DNA Separation Buffer	Bioptic	C104406	DNA fragment analyzer supporting products
E. coli derived ultrapure LPS (serotype0111: B4)	Sigma-Aldrich	Cat#L-2630
DNA Quantitative Reagent	vazyme	EQ111	DNA Quantitative Reagentt is a simple, sensitive and accurate double-stranded DNA (dsDNA) fluorescence quantitative assay kit.
Erythrocyte lysis buffer (10x)	BioLegend	Cat#420301
Fetal bovine serum	Gibco	Cat#10099141C
MS Separation columns	miltenyi	130-042-201	MS Columns are designed for positive selection of cells.
RPMI 1640 medium	Gibco	C11875500BT
S2 Gel electrophoresis needle	Bioptic	C105101	DNA fragment analyzer supporting products
Surgical instruments: scalpel, dissection scissors, microdissection scissors, fine forceps, blunt anatomical forceps, needle holder	Fisher Scientific	https://www.fishersci.com/us/en/browse/90184247/surgical-tools	Can be purchased from other qualified suppliers
Syringe (1 mL)	Fisher Scientific	https://www.fishersci.com/shop/products/sterile-syringes-single-use-12/14955464	Can be purchased from other qualified suppliers
TruePrepTM DNA Library Prep Kit V2 for Illumina	vazyme	TD501-01	TruePrep DNA Library Prep Kit V2 for Illumina is a purpose-built kit specifically developed for Illumina's high-throughput sequencing platform. Using the kit, DNA can be prepared into sequencing libraries dedicated to Illumina's high-throughput sequencing platform.
TruePrepTM Index Kit V2 for Illumina	vazyme	TD202	TruePrep Index Kit V2 for Illumina is a Kit for the TruePrep DNA Library Prep Kit V2 for Illumina. It can be used to prepare 96 different double-ended Index libraries.