GSM6416064: Biliary tract cancer, tumor 1, scRNAseq; Homo sapiens; RNA-Seq
Tumor samples were digested in RPMI containing Collagenase I & II and DNAse I, minced, and digested for one hour using the GentleMACS system. Isolation of live cells was performed using MACS LS columns (Miltenyi Biotec). Library was performed according to the manufacter's instructions (single cell 10x 5' version 1 protocol, 10x Genomics). Briefly, cell suspensions were combined with master mix and loaded into the chip with gel beads to generate the gel bead-in-emulsion (GEM), which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. Reverse transcription was performed on the poly-A RNA within each GEM. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads and amplified by PCR. To prepare libraries from cDNA, SPRIselect beads were used to isolate the correct size fragments. Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added during library construction.