GSM8702823: CRISPRko, transcriptomes scRNA-seq, rep3; Homo sapiens; RNA-Seq
Adherent cells were digested with 0.25% trypsin and collected in a 15mL tube containing serum-containing medium, followed by centrifugation and washing of the cell pellet with serum-free basal medium to obtain a single-cell suspension. The library construction protocol for single-cell RNA sequencing (scRNA-seq) was performed following the manufacturer's instructions using the 10x Genomics Chromium Single Cell 3' Library reagents V3. The protocol involved the generation of gel bead-in-emulsion (GEM) using cells resuspended in a master mix along with partitioning oil and gel beads. The GEMs encapsulated individual cells' poly-A RNA, which was retrotranscribed into cDNA containing an Illumina R1 primer sequence, Unique Molecular Identifier (UMI), and the 10x Barcode. The barcoded cDNA was then subjected to various steps, including cleaning up with Silane DynaBeads, PCR amplification, sgRNA enrichment PCR, and fragmentation of the remaining amplified cDNA. Appropriately sized fragments were selected using SPRIselect reagent. The library was then prepared by ligating paired-end constructs with P5 and P7 sequences and a sample index. Finally, the enriched sgRNA library and fragmented cDNA were sequenced on an Illumina NovaSeq6000 PE150 platform, following the standard 10x libraries configuration.