GSM8641729: pooled cell lines, Multi-Seq; Homo sapiens; OTHER
ells were prepared for single-cell RNA sequencing by trypsin digestion for 10 minutes at 37C when harvested from in-vitro culture. For harvest of organoids and in-vivo xenograft tumors, cells were harvested by resection, chopping with a razor blade into a slurry, and then resuspension in 1 mg/mL Liberase TM. Suspensions were incubated shaking at 37C at 150 RPM for 45 minutes after which cells were resuspended in ACK buffer and incubated at room temperature for 10 minutes. Resuspension in 1X PBS was performed two times following this for wash and removal of digestion buffers. Cells were passed through a 70 micron mesh filter to arrive at single-cell suspensions. Cell suspensions were then hashed using HTO oligos or Totalseq-A antibodies. Cell suspensions were assigned unique oligo-HTO or antibody-HTOs at the point of experimentation. Cells were incubated with corresponding HTO conjugate for 20 minutes on ice. Cells were then washed 3X in 1X cold PBS and then recounted. Cells were then pooled together from multiple hashed suspensions such that there were equivalent numbers of cells from each suspension prior to 10X single-cell preparation. All single-cell studies were hashed in this way.. GENEVA libraries were prepared according to single-cell RNA sequencing Chromium kit instructions. Modifications were performed after the cDNA step in which the small fractions were harvested and then amplified in separate PCR reactions for enrichment of the hashtag reads, also as specified in Chromium kit. Sequencing was performed for the transcriptome and hashtag libraries at a 95%/5% ratio (transcriptome/hashtag). Sequencing targets were 25,000 reads/cell based on expected cell counts determined by number of cells loaded at input into the chromium kit.