GSM8463925: OSKM, replicate 1, snRNAseq; Mus musculus; RNA-Seq
Prepare nuclei using a singulator 100 system (S2 Genomics). The singulator 100 system is designed to automatically process solid tissues into suspensions of nuclei using mechanical disruption coupled with chemical dissociation in a single-use cartridge. A mass of 20~100mg of tissue is recommended. Determine the nuclei concentration using a LUNA-FL™ Automated Fluorescence Cell Counter (logos biosystems) and examine the nuclei morphology using microscopy. Libraries were prepared using the Chromium controller according to the 10x chromium Next GEM Single Cell 3' v3.1 protocol(CG000315). Briefly, the cell suspensions were diluted in nuclease-free water to achieve a targeted cell count of 10,000. Cell suspension was mixed with master mix and loaded with Single Cell 3'V3.1 Gel Beads and Partitioning Oil into a chromium Next GEM chip G. RNA transcripts from single cells were uniquely barcoded and reverse transcribed within droplets.cDNA molecules were pooled and the cDNA pool then go through an end repair process, the  addition of a single 'A' base, and then ligation of the adapters. The products are then purified andenriched with PCR to create the final cDNA library. The purified libraries were quantified using    qPCR according to the qPCR Quantification Protocol Guide(KAPA) and qualified using the Agilent Technologies 4200 TapeStation(Agilent technologies). And then the libraries were sequenced usingNovaSeq platform(Illumina) according to the read length in the user guide. LibrarieswerepreparedusingtheChromiumcontrolleraccordingtothe10xchromium Next GEM SingleCell3'v3.1protocol(CG000315).Briefly,thecellsuspensionsweredilutedinnuclease‑free watertoachieveatargetedcellcountof10,000.Cellsuspensionwasmixedwithmastermixand loadedwithSingleCell3'V3.1GelBeadsandPartitioningOilintoachromium Next GEM chip G.RNAtranscriptsfromsinglecellswereuniquelybarcodedandreverse‑transcribedwithindroplets.cDNAmoleculeswerepooledandthecDNApoolthengothroughanendrepairprocess,the additionofasingle'A'base,andthenligationoftheadapters.TheproductsarethenpurifiedandenrichedwithPCRtocreatethefinalcDNAlibrary.Thepurifiedlibrarieswerequantifiedusing   qPCRaccordingtotheqPCRQuantificationProtocolGuide(KAPA)andqualifiedusingtheAgilentTechnologies4200TapeStation .