GSM8008711: WT, replicate 2, scRNA-seq; Mus musculus; RNA-Seq
Bone marrow cells were isolated from the tibia and femur by flushing a 25G syringe with 1X PBS containing 2% FBS. The red blood cells were removed using ACK lysis buffer before antibody incubation. The cells were then counted and incubated with antibodies. The linage-marker-negative (Lin-) c-kit+ population containing hematopoietic stem and progenitor cells was sorted Sorted single-cell suspension, 10x barcoded gel beads and oil (10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1 PN 1000268) were loaded into 10x Chromium™ Single Cell Chip to capture single cells into nanoliter-scale oil droplets by 10xChromium X Controller and generate Gel Bead-In-EMulsions (GEMs). cDNA from single cells were synthesized and barcoded by incubation of the GEMs in a thermocycler machine and then purified from GEMs by DynaBeads (10xGenomics PN 2000048). cDNA was pre-amplified by PCR to generate sufficient mass for sequencing library construction. The single-cell cDNA libraries were constructed by following 10xGenomics 3' NextGem 3.1 version kit instructions. The final constructed 3'-biased single-cell libraries were sequenced by the Illumina Novaseq6000 machine, targeting 50,000 read pairs/cell.