title: GSM6283027: UCB+OP9, non-adherent, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: After 24 h culture, adherent(S2) and non-adherent cells(S3) were subjected to scRNA-seq. For the control, we used crude RBC depleted UCB(S1). Single-cell suspension were processed through the 10x Genomics Chromium Controller following the protocol outlined in the Chromium Single Cell 3' Reagent Kits User Guide. Chromium Next GEM Single Cell 3' Kit v3.1 (Cat# PN-1000269), Chromium Next GEM Chip G Single Cell Kit (Cat# PN-1000127) and Dual Index Kit TT Set A (Cat# PN-1000215) were applied during the process.Briefly, UCB cells with/without OP9 pre-conditioning underwent single-cell capture procedure. Single-cell suspensions containing approximately 16,500 live cells per sample were loaded into Chromium controller to generate 10,000 single-cell gel-bead emulsions for library preparation and sequencing. Oil droplets of encapsulated single cells and barcoded beads (GEMs) were subsequently reverse-transcribed in a Veriti Thermal Cycler (Thermo Fisher Scientific), resulting in cDNA tagged with a cell barcode and unique molecular index (UMI). Then cDNA was amplified to generate single-cell libraries according to the manufacturer's protocol. Quantification was made with an Agilent Bioanalyzer High Sensitivity DNA assay (Agilent,High-Sensitivity DNA Kit, Cat# 5067-4626). Subsequently amplified cDNA was enzymatically fragmented, end-repaired, and polyA tagged. Cleanup/size selection was performed on amplified cDNA using SPRIselect magnetic beads (Beckman-Coulter, SPRIselect, Cat# B23317). Next, Illumina sequencing adapters were ligated to the size-selected fragments and cleaned up using SPRIselect magnetic beads. Finally, sample indices were selected and amplified, followed by a double-sided size selection using SPRIselect magnetic beads. Final library quality was assessed using an Agilent Bioanalyzer High Sensitivity DNA assay. Samples were then sequenced on an Illumina NovaSeq 6000 as paired-end mode (read1: 28bp; read2: 91bp).