GSM8578514: Signalling perturbations library 3, BC; Homo sapiens; RNA-Seq
Cells were dissociated with EDTA/Trypsin. For hashtagged samples, 1e6 cells were stained with their corresponding TotalSeq-A anti-human Hashtag antibody (see HTO antibody details above). Cells were sorted for viability using propidium iodide before being pooled for 10X Genomics Chromium Single Cell 3' V3 capture, targeting 2e4 cells. mRNA libraries were constructed according to manufacturer's instructions (Chromium Single Cell 3' V3). The barcoding (BC) libraries were generated from a portion of the cleaned up full length amplified cDNA from each mRNA sample pool. In brief, the cDNA regions containing the barcode cassette were amplified and partial P5 and P7 sequencing adaptors were appended in one round of PCR. After clean-up to remove primers, additional PCR was performed as per the SI-PCR protocol in the Chromium gene expression library construction workflow  with a unique i7 indexed R primer from Chromium i7 Multiplex Kit for a total of 14 cycles. For more details, see methods. The HTO libraries were generated following additional methods from Stoeckius et al., (2018) to capture the fraction of droplets containg HTO-derived cDNA and generate the hashing libraries.