GSM4059983: Keratinocyte-cocultured CVG1 with shpanT; Homo sapiens; RNA-Seq
To prepare cDNAs for single cell transcriptome analysis, Single nuclei are stained using trypan blue for visualization, counting and viability assessment using the Countess II Automated Cell Counter and diluted as needed to a final concentration of 700-1200 nuclei/μL in 1x PBS + 0.04% BSA.  A minimum of two reproducible counts with SD <25% are required before droplet preparation and <5% of input should be viable cells. Nuclei are added to a master mix containing reverse transcription reagents and primer, and are then transferred to the ChromiumChip with gel beads and partitioning oil for the preparation of nanoliter-scale Gel Bead-In-Emulsions (GEMs). The emulsion undergoes reverse transcription to produce cDNA with a cellular 10X barcode and UMI, and is recovered with Dynabeads MyOne SILANE magnetic beads according to the manufacturer's directions.  Subsequently, the cDNA undergoes 8-14 cycles of amplification before clean-up using SPRIselect Reagent (Beckman).  Total cDNA yield is calculated following a quality assessment (High Sensitivity D5000 ScreenTape, Agilent) and fluorometric quantitation (Qubit). Libraries are constructed by enzymatic fragmentation, end repair, and A-tailing according to the manufacturer's instructions.  A double-sided SPRIselect Reagent (Beckman) clean up prepares samples for adapter ligation and another round of magnetic bead clean-up before sample indexing PCR for 5-16 cycles, depending on the cDNA input for library construction. A further double-sided size selection produces libraries of 400-600bp, which are quantified for paired-end sequencing (26bp x 98bp) using a NextSeq 500 System.
GEO Accession
GSM4059983