GSM8889621: CD27_low_rep4; Mus musculus; ATAC-seq
Bone marrow (BM) cells were flushed from the tibiae, femora, and pelvis. Cells were then stained with anti-c-Kit microbeads (catalog no. 130-091-224, Miltenyi Biotech) and c-Kit+ cells were enriched by columns. These cells were stained with CD201 (eBio1560), CD150 (TC15-12F12.2), CD48 (HM48-1), Sca-1 (D7), c-Kit(2B8), CD27 (LG.7F9) and with a lineage-marker cocktail of B220 (RA3-6B2), Gr-1 (RB6-8C5), TER-119 (TER-119). Cells were sorted by FACSAria III cell sorter (Becton Dickinson, US) according to surface markers expression under the single-cell sorting mode. The Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme, TD711) was used to ATAC-seq library preparation. CD27- or CD27+ ESALAMLSK cells were lysed with a buffer containing RS Buffer, 10% NP40, 10% Tween20, and 1% Digitonin. Chromatin was fragmented using a Tagmentation Mix (TW Buffer, 10% Tween20, 1% Digitonin, nuclease-free ddH2O, 5× TTBL, and TTE Mix V50) by incubated at 37°C for 30 minutes. Reactions were stopped with Stop Buffer, and then the nucleosome-free DNA fragments were extracted using DNA Extract Beads. The extracted DNA fragments were amplified via PCR with 2× CAM and indexed primers (N5XX and N7XX) under the following conditions: 72°C for 3 minutes, 95°C for 3 minutes, followed by 10–21 cycles of 98°C for 10 seconds, 60°C for 5 seconds, and 72°C for 1 minute. Size-selection of PCR products were conducted by using DNA Clean Beads (0.55× and 1.2× ratios) and finally sequenced on an Illumina NovaSeq™ X Plus.