GSM8223015: G1M6 Gene Expression; Mus musculus; RNA-Seq
Single cell RNA-Seq library prep was provided by the Single Cell Core at Harvard Medical School, Boston, MA. For each library, cells were resuspended in DPBS with 0.04% FBS to achieve a target concentration of 200–1500 cells per microliter. Samples with more than 85% viability were processed. For each library, ~16,000 cells were loaded onto a 10× chromium controller to target a recovery of 10,000 cells. Both gene expression and TCR libraries using the Chromium Next GEM Single Cell 5′ Reagent Kits (v2) were prepared following the 10X Genomics' user guide. Briefly, the single cells were encapsulated and barcoded within nanoliter-sized droplets along with the reagents necessary for cell lysis and reverse transcription. Within the droplet cells are lysed and the mRNA molecules are captured and barcoded during reverse transcription. Subsequently, full-length cDNA was made, followed by enzymatic fragmentation. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The libraries were sequenced at the Biopolymers Facility at Harvard Medical School using a NovaSeq 6000 V1.5 S4 full flow cell following sequencing guideline recommended by 10X Genomics protocol.