Single cell methylation of dormant and active hematopoietic stem cell and multipotent progenitor from H2B-GFP bone marrow after 100 days of chase
Single cell methylation of dormant and active hematopoietic stem cell and multipotent progenitor from H2B-GFP bone marrow after 100 days of chase
Scl-tTA; H2B-GFP mice were treated with doxycycline for 100 days, which turn off constitutively expressed H2B-GFP, thus allow the marking of H2B-GFP+ dormant cells and H2B-GFP- active cell. Bone marrow was isolated after 100 days of chase, red cell lysed and lineage depleted 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), APC-Cy7 anti-mouse Sca1 (clone D7; BD Biosciences; 560654), PE anti-mouse EPCR (clone eBio1560; eBioscience; 12-2012-82), 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), 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). Index sorting was performed to sort single CD48– CD135- LSK cells into 384-well plate containing 2 µL RLP plus buffer (Qiagen, Cat #1053393) and 1 µL water per well and immediately frozen at -80 °C.  single cells were lysed in 2 µL RLP plus buffer (Qiagen, Cat #1053393) and 1 µL water per well and immediately frozen at -80 °C.  Single-cell methylome and transcriptome sequencing (scM&T-seq) libraries were generated based on the high-throughput protocol published previously (Cerrizuela et al., 2022) with the modification that no GpC methyltransferase incubation was done and that 384 cell could be multiplexed for the sequencing of the methylation layer. 