GSM6657360: gRNA enrichment library; Homo sapiens; RNA-Seq
Cells were harvested and processed following the 10x Genomics demonstrated protocol for the preparation of single cell suspensions. See also 10x Genomics V3 Single Cell 3' Solution kit user guide. For the sgRNA enrichment libraries, briefly, 10% of the full length 10X cDNA was used as template for two consecutive PCRs aimed at enrichment of gRNA cassette and addition of sequencing adapters with Hifi HotStart ReadyMix (Roche, 7958935001). For CROPseq library: 5000 cells were processed with Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead v3.1 kit (10X Genomics, 1000128) following the manufacturer's instructions. For gRNA library: 10% of cDNA was used for sgRNA enrichment and creation of a specific gRNA library. For higher resolution and better cell assignments, gRNA sequences were amplified with Hifi HotStart ReadyMix (Roche, 7958935001) and gRNA cassette specific primers. gRNA cassette maps to CROPseq Guide-Puro vector (addgene plasmid #86708). Both libraries were sequenced on Illumina NovaSeq6000 with 28 cycles for read 1 and 91 cycles for read 2.