GSM5532516: WBM_2c; Mus musculus; RNA-Seq
Bone marrow cells were isolated from the spine, hips, femur and tibia of mice. A single-cell suspension was generated after red blood cell lysis ACK buffer. Cells were tagged with cell hashing (Stoeckius et al, 2018) oligo-tagged antibodies (BioLegend) according to manufacturer's instructions and pooled on a 10x Chromium instrument (10x Genomics) to generate barcoded single-cell beads-in emulsion (GEM) according to the manufacturer's protocol. scRNA-seq libraries were prepared using the Chromium Single-Cell' v3 Reagent Kit (10x Genomics) following the direction of the manufacturer. The libraries were quantified using Qubit fluorometric quantification (Thermo Fisher Scientific). The quality was assessed on an Agilent Bioanalyzer 2000, obtaining an average library size of 455 bp. Libraries were pooled and normalized to 2 nmol/L and clustered on a HiSeq4000 high output mode on a paired-end read flow cell and sequenced for 28 cycles on R1 (10x barcode and the UMIs). Followed by 8 cycles of i7 index (sample index), and 98 bases on R2 (transcript), with a coverage around 130M reads per sample.    Single Cell 3' Reagent Kits: Chromium™ Single Cell 3' Library & Gel Bead Kit v3, PN-1000157; Single Cell 3' Chip Kit v3 PN-1000123 and i7 Multiplex Kit PN-220103(10x Genomics). Samples were tagged with cell hashing oligo-tagged antibodies (BioLegend) according to manufacturer's instructions.