title: GSM6805267: Wild-type LSKs, derived from CreER+ Stat3f/f-containing BM chimera [SKO-Com]; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow (BM) cells were flushed from femoral and tibial bones of chimeric mice into Dulbecco's Minimal Essential Medium (DMEM) supplemented with 1% fetal bovine serum. Single cell suspensions of BM cells were treated with red blood cell lysis buffer (Tonbo Bioscience) and incubated with biotinylated antibodies against hematopoietic lineage markers (CD11b, Gr1, CD3, B220, Ter119; Thermo Scientific) for 30 mins. BM cells were then washed with 1X phosphate buffered saline (PBS) and incubated with goat anti-rat IgG microbeads (Miltenyi Biotec). After 30 mins of incubation, the cells were washed in PBS and passed though magnetic-activated cell sorting (MACS) columns (Miltenyi Biotec) to purifiy Lineage- hematopoietic stem and progenitor cells (HSPCs) by negative selection. Lineage- HSPCs were then subjected to FACS to purify Lin-ckit+Sca1+ cells (LSKs). The following LSKs were purified: CD45.2+ CreER+ and CD45.1+ CD45.2+ wild type competitors from control mice containing a 1:1 ratio of CreER+ and wild type competitor cells; and CD45.2+ CreER+ Stat3f/f and CD45.1+ CD45.2+ wild type competitors from test mice containing a 1:1 ratio of CreER+ Stat3f/f and wild type competitor cells. LSKs were purified 8 weeks following tamoxifen treatment to induce Stat3 gene deletion in CreER+ Stat3f/f cells. Single cell suspensions of LSKs (1000 cells/mL) were prepared in PBS supplemented with 0.04% bovine serum albumin (BSA) and used for single cell RNA sequencing. Purified LSKs were loaded onto a Chromium Single Cell Chip (10X Genomics, Pleasanton, CA) and processed for 3' single cell RNA seq libraries. The cells were lysed and barcoded for reverse transcription at a target capture rate of 10000 single cells per sample. Bar coded libraries were pooled and sequenced using Novoseq 6000 SP 100-cycle flow cell (Illumina, CA) to an average depth of 100,000 reads per cell.