GSM9032841: H358 cells labeled by Lib3b +Vehicle(DMSO); Homo sapiens; RNA-Seq
Single-cell suspensions were prepared and assessed for viability (>90%) using trypan blue staining by TC20 Automated Cell Counter (BioRad, Inc). Viable cells were processed with a modified protocol of Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics , Inc). (incorporating Feature Barcoding technology for CRISPR) Gel beads-in-emulsion (GEMs) were created to capture ~8,000 cells per channel. After GEM reverse transcription and cDNA amplification, a 0.6× size selection was used to collect larger cDNA fragments for gene expression library construction. In contrast, a 0.6–1.2× fraction containing shorter cDNAs was dedicated to generating gRNA barcode libraries. Targeted PCR amplification further enriched gRNA barcode transcripts with primers specific to gRNA sequences. To ensure sufficient coverage and minimize amplification bias, each cDNA sample underwent 3 parallel amplification reactions for both BC1 and BC2. Libraries were indexed using the Dual Index Kit TT Set A (10x Genomics, Inc) and sequenced on an Illumina NovaSeq X Plus system