GSM9414679: Tet2 Coldspot Week1 biological rep 2; Mus musculus; RNA-Seq
Isolated mouse calvaria bone buttons were submerged in 5% HSC buffer (Gemini Bio, Cat. #900-208) on ice before further processing and sorting. To harvest bone marrow cells, bone pieces from each group were placed in 1mL of FACS buffer, grinded, and passed through a 70 mm cell strainer. The remaining bone pieces were transferred into a 15 mL conical tube for enzymatic digestion. Cells were centrifuged at 460g for 7 minutes at 4°C and resuspended in 0.5 mL of HSC buffer for FACS. For enzymatic digestion, 2 mL digestion buffer containing 1x HBSS (Gibco, Cat. #14175095), 1mg/mL collagenase IV (Gibco, Cat. #17104019), 20 μL of DNase (1U/μL, ThermoFisher Cat. #EN0521), and 4 mg of Dispase (ThermoFisher, Cat. #17105041) was used to resuspend the bone fragments (0.5mL per sample) and incubated in 37°C for 30 minutes. The samples were then rinsed with 5% FACS buffer, resuspended and passed through the 70 mm cell strainer. The digested sample was then palleted, resuspended in 100-200 μL of HSC buffer and combined with the undigested counterpart of cells. Cell sorter was used to isolate GFP+ mutant cells and GFP- microenvironment cells (BD FACS Aria III sorter) for scRNA seq (10X Genomics). Cell suspensions were processed to generate single-cell RNA-Seq libraries using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per the manufacturer's recommendations, as summarized below. Samples were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs (Gel Bead-in-Emulsions). GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, the pooled GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, ThermoFisher Scientific). The purified cDNA was further amplified by PCR to generate sufficient material for library construction.  Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by end repair, A-tailing, adaptor ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Sequence data was generated using Illumina's NovaSeq 6000.