GSM8668994: reprogram_reads; Homo sapiens; RNA-Seq
Adherent cells were dissociated using 1x TrypLE Express Enzyme (Gibco #12604-021) Full-length barcoded cDNA amplicons were obtained from single cells using the 10X Genomics Next GEM Single Cell 3' Kit V3.1. Briefly, 20,000 sorted CD34+ cells and 2,000 control fibroblasts were loaded onto the 10X Genomics Chromium Controller (X) to partition single cells into Gel Beads-In Emulsion (GEMs). cDNA was generated from polyadenylated mRNA transcripts, incorporating a unique molecular identifier (UMI) and a cell-specific barcode for each transcript. Barcoded cDNA was pooled, purified with SILANE magnetic beads (10X Genomics #PN-2000048), and amplified by PCR. Barcoded cDNA libraries were prepared for sequencing according to the Oxford Nanopore Technologies (ONT) 3' cDNA protocols. Briefly, cDNA amplicons were biotin tagged by PCR with the primers shown in the ONT protocols. Biotinylated cDNA was purified with Dynabeads™ M-280 Streptavidin beads (Invitrogen #11205D), followed by secondary PCR amplification and Agencourt AMPure XP bead clean-up (Beckman Coulter #A63881). Products were assessed for quality following ONT recommendations. Long-read cDNA libraries were prepared for sequencing as in ONT protocols and sequenced on an ONT GridION or PromethION Solo 2 (P2) sequencer. Raw reads were base-called with guppy using the High Accuracy basecalling model and stored as fastq files.