ATAC-seq of mouse hematopoietic stem cell and multipotent progenitor cell populations
ATAC-seq of mouse hematopoietic stem cell and multipotent progenitor cell populations
To prepare for BM cell suspension, bones were crushed and incubated with ACK buffer for 5 minutes at room temperature to remove excess red blood cells. Lineage depletion was performed using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL technologies, #19856) according to the manufacturer's instruction. The Lin-depleted cells were then stained with the following antibodies: BV711 anti-mouse c-Kit (clone 2B8; BioLegend; 105835) or BUV395 anti-mouse c-Kit (clone 2B8; BD Biosciences; 564011), APC-Cy7 anti-mouse Sca1 (clone D7; BD Biosciences; 560654), PE anti-mouse EPCR (clone eBio1560; eBioscience; 12-2012-82), FITC anti-mouse CD34 (clone RAM34; eBioscience; 11-0341-82) or eFluor 660 anti-mouse CD34 (clone RAM34; eBioscience; 50-0341-82), PE-Cy7 anti-mouse CD48 (clone HM48-1; BioLegend; 103424), BV421 anti-mouse CD135 (clone A2F10.1; BD Biosciences; 562898) or APC anti-mouse CD135 (clone A2F10; BioLegend; 135310),PE-Cy5 anti-mouse CD150 (clone TC15-12F12.2; BioLegend; 115912) or BV785 anti-mouse CD150 (clone TC15-12F12.2; BioLegend; 115937), BUV496 anti-mouse CD16/32 (clone Ab93; BD Biosciences; 751694), Alexa Fluor 700 anti-mouse CD4 (clone GK1.5; eBioscience; 56-0041-82),Alexa Fluor 700 anti-mouse CD8a (clone 53-6.7; eBioscience; 56-0081-82), Alexa Fluor 700 anti-mouse CD11b (clone M1/70; eBioscience; 56-0112-82),Alexa Fluor 700 anti-mouse GR1 (clone RB6-8C5; eBioscience; 56-5931-82), Alexa Fluor 700 anti-mouse B220 (clone RA3-6B2; eBioscience; 56-0452-82), Alexa Fluor 700 anti-mouse Ter119 (clone TER-119; BioLegend; 116220).  All antibody staining were performed at 4 °C for approximate 30 minutes. Cells were then washed to remove unbound antibodies and resuspended in FACS buffer containing 7-AAD (BD Biosciences; 51-68981E) prior to cell sorting using BD FACSAria-II/III, BD Fusion or BD Symphony S6 (BD Biosciences). Between 300 – 3000 cells from each HSPC populations were sorted. cells were sorted, pelleted and resuspended in cold lysis buffer, followed by incubation on ice for three minutes to release nuclei. Nuclei were then washed in ATAC-Resuspension Buffer (RSB) containing 0.1% Tween-20, pelleted, and resuspended in transposition buffer containing Tn5 transposase. Transposition reactions were performed via incubation at 37 °C in a thermomixer with shaking at 1,000 rpm for 30 minutes. DNA was purified using AMPure XP magnetic beads and libraries were amplified by qPCR using NEBNext High-Fidelity Master Mix and custom i5 and i7 primers.