GSM9514432: BMD STING-seq pilot GDO 1; Homo sapiens; OTHER
We performed single-cell RNA-seq using 10X Genomics Chromium Next GEM Single Cell 5′ Reagent Kits v2 (dual index) on cells detached from culture plates. We labeled cells from each state with different hashtag antibodies targeting cell surface proteins (BioLegend TotalSeq 394661, 394663, 394665, 394667) following the manufacturer's recommendations. We then pooled equal numbers of cells from each state into a single sample and aliquoted them into as many reactions as needed to capture approximately 200,000 cells in total. We used 2 microfluidics channels for the pilot experiment and 6 channels for the full experiment. We constructed sequencing libraries for the three different barcoded nucleic acids for each reaction according to 10X Genomics protocols and pooled them disproportionately so that mRNA-derived cDNA libraries represented ~90% of the sample, with gRNA and hashtag libraries comprising the remaining 10%. We sequenced the pooled library to a depth of approximately 20,000 mRNA/cDNA reads per cell on an Illumina NextSeq 500 sequencing platform for the pilot experiment and on an Illumina NovaSeq X for the full experiment.