GSM7232964: Trmt6 cKO, scRNA-seq; Mus musculus; RNA-Seq
BM cells were harvested by crushing the bones (Femur, Tibia and Hip). Then, red blood cells were lysed with Ammonium-Chloride-Potassium (ACK) buffer and the remainning cells were washed twice with PBS, lineage depleted using the Lineage Cell Depletion Kit (Miltenyi), labelled with the following antibody cocktail: anti-cKit, anti Sca-1 and anti Flt3 antibodies and FACS sorted   FACS purified HSPCs were loaded 30 min after the sorting onto a Chromium Single Cell Chip and processed with the Chromium Controller (10x Genomics) according to the manufacturer's instructions for single cell barcoding at a target capture rate of 4000 individual cells per sample. Libraries were prepared using Chromium Single-Cell 3′ Reagent Kits v2 (10x Genomics)  single cell RNAseq library using Chromium Single-Cell 3′ Reagent Kits v2 (10x Genomics) Library was performed according to the manufacter's instructions (single cell 3' protocol, 10x Genomics). Briefly, HSPCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.