GSM6435433: Dialout_2kG_2PMDox1_5; Homo sapiens; OTHER
Cells were "superloaded" at 150,000 cells per 10X Chromium controller lane, on 20 lanes (for a total of 3 million cells), and scRNA-seq and dialout libraries prepared as described below. 1) scRNA-seq libraries were prepared using 3' scRNA-seq V3 kits (10X Genomics), according to manufacturer instructions. Note that the 10x Indexes were a mix of 4 oligos for each library (each starting with a different base to avoid sequencing biases). Thus each scRNAseq library has 4 fastq files (denoted _a, b, c & d). 2) From the initial amplified cDNA, we used a two stage PCR protocol to generate “dialout” libraries, for each lane. Because the CROP-seq vector expresses a PolII polyadenylated transcript that ends just downstream of the guide sequence, the dialout libraries identify the sgRNA sequences associated with each droplet. In several cases multiple libraries were made for a sample. Libraries without ".2" in their name (e.g. Dialout_2kg_11AMDox_4_ATTACTCG) were sequenced on an Illumina NextSeq machine. Others, e.g. "Dialout_2kg_11AMDox_6.2_CGCTCATT" were sequenced on an Illumina Novaseq machine, together with scRNA-seq libraries.