title: GSM6436806: SK-N-DZ, lane 1, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Ten days after transduction, puromycin-selected cells were washed twice with PBS and dissociated with accutase (37° C, 5 min). Cells were collected in PBS + 0.04 % BSA, counted and diluted to 10^6 cells/ml Single-cell libraries were constructed following the manufacter's instructions (10x Genomics, Single Cell Gene Expression v3.1 protocol). Briefly, 20,000 SK-N-DZ cells per lane were resuspended in the master mix and loaded alongside the gel beads and the partitioning oil into the chip (G) to generate the GEMs. Polyadenylated mRNA in every single GEM was reverse-transcribed to cDNA with a oligo(dT) primer, that contains the illumina TruSeq Read 1 primer sequence, the 10x Barcode and Unique Molecular Identifiers (UMIs). The barcoded cDNA was pooled and cleaned up with Silane DynaBeads. Full-legnth cDNA was amplified by PCR. AFter enzymatic fragmentation, end repair, A-tailing and ligation, the apropiated sized fragments were selected with SPRIselect beads for subsequent library construction. During the final library construction, the Illumina TruSeq Read 2 primer sequence was introduced alongside a sample index (four per lane). Forty nanogram of full-length cDNA was used as input for the construction of gRNA enrichment libraries following a previously published hemi-nested PCR strategy (Hill et al., 2018)