GSM7898119: P8_BCa_scRNAseq; Homo sapiens; RNA-Seq
Fresh bladder cancer tissues were obtained from patients and washed with ice-cold PBS. Then, the tissues were subjected to prepare single cell suspensions with tumor dissociation kit. Subsequently, the cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Upon dissolution of the Gel Bead in a GEM, primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence were released and mixed with cell lysate and Master Mix. Barcoded, full-length cDNAs were then reverse-transcribed from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. The Single Cell 3' Protocol produced Illumina-ready sequencing libraries. A Single Cell 3' Library comprised standard Illumina paired-end constructs which begin and end with P5 and P7. The Single Cell 3' 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1, while Read 2 was used to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 were standard Illumina® sequencing primer sites used in paired-end sequencing.