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 extra....

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 ref.......

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.