GSM5693897: LK WT_batch2; Mus musculus; RNA-Seq
Mouse BM cells were isolated from pooled femora, tibiae and ilia bones, by crushing them in cold-PBS-0.04%BSA. For progenitor cell sorting, bone marrow cells were incubated with a biotin-antibody cocktail (CD3, B220, CD11b, Gr1, Ter119), followed by magnetic depletion with streptavidin-conjugated magnetic nanobeads (BioLegend). LK (Lin-cKit+) and LSK (Lin-Sca+cKit+) cells were sorted with a FACS Fusion II instrument (BD Biosciences). An equal number of FACS sorted LK (Lin-cKit+) and LSK (Lin-Sca1+cKit+) were combined from a female mouse and from a male mouse of each genotype, resulting in a pool of ~24,000 input cells. The cells was processed on a 10X Genomics Chromium controller to generate a cDNA library, according to manufacturer's protocol. Sequencing libraries were generated from cleaned, amplified cDNA using the Chromium Next GEM Single Cell 3' Library kit (10X Genomics), with version 3.1 chemistry, including reagents for fragmentation, adaptor ligation, and sample index PCR. Single-cell RNA-seq paired-end 100 bp reads were generated using the Illumina NovaSeq 6000 system, at a depth of ~30,000 paired-reads per cell.