GSM6502787: Bonemarrow Progenitors ATTO High replicate2; Mus musculus; RNA-Seq
Mouse bone marrow HSPs and precursor neutrophils were enriched by two step negative selection. Initially Ly6G+ cells were depleted, followed by further depletion with Ly6G, CD3, B220, Ter119, CD115, F4/80, Siglec-F, and CD11C.  Lineage depleted cells enriched for HSPs and neutrophil precursors were electroporated with Cas9-sgRNA RNP complexes against NIPBL using Lonza 4D-NucleofectorTM X unit (DN-107 program). Cells were cultured for 72hrs in opti-MEM medium supplemented with 10%FBS, glutamine, penicillin and streptomycin, β-mercapto ethanol, 25ng/mL mSCF, 25ng/mL mFLT3. ATTO high and ATTO lo/negative cells were FACS sorted and were used for single cell RNA sequencing. Library was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell HT Training Reagent Kit, 10x Genomics).