GSM7143098: Old_ATAC-seq, sample6; Mus musculus; ATAC-seq
Bone marrow cells were harvested by flushing the femur, tibia, humerus, and pelvis bones. Lineage negative (Lin-) compartment was enriched by using Streptavidin Microbeads with biotinylated antibodies (B220, TCRβ, NK1.1, CD11b, Gr-1, and Ter119). Collected Lin- cells were stained with c-Kit, Sca-1, CD150, and CD48 to define and sort HSCs. ~33,000 Lin -Sca-1 +c-Kit +CD150 +CD48 -HSCs were sorted and pooled, and nuclei isolation was performed according to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (GEX) protocol (10x Genomics). scRNA-seq and scATAC-Seq libraries were constructed using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kit (10x Genomics, PN-10000285) according to the manufacturer's protocol. Briefly, 5-7K nuclei in 1x nuclei buffer were loaded with a target output of∼4,000. Single nuclei were encapsulated into emulsion droplets using Chromium Controller (10x Genomics). Transposition, reverse transcription and library preparation were performed on C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad). Amplified cDNA was evaluated on a Agilent BioAnalyzer 2100 using a High Sensitivity DNA Kit (Agilent Technologies, PN5067-4626) and final libraries on a Agilent TapeStation 4200 using High Sensitivity D1000 ScreenTape (Agilent Technologies,PN5067-5584). Individual libraries were normalized to 2nM and pooled for sequencing. Pools were sequenced with 200 cycle run kits (50bp Read1, 10bp Index1 24bp Index2 and 90bp Read2) on the NovaSeq 6000 Sequencing System (Illumina) and analyzed using CellRanger software.