GSM9108078: H25919-08; Homo sapiens; RNA-Seq
For scRNAseq of organoids: cell dissociation using papain/DNAse solution, with incubation for 60min for organoids. Fixed snRNAseq: Sample preparation for deparafinization and isolation of cells from FFPE tissue sections following 10x Genomics for Fixed RNA seq - protocol CGO000632_RevB, using gentleMACS Octo dissociator with heaters (Miltenyi, Cat n. 140-004-386.06). For scRNAseq of organoids: Approximately 10,000 vital single-cells of each sample were processed for single-cell RNA sequencing using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1, the Chromium Next GEM Chip G and the Single Index Kit T Set A (10X Genomics) according to the manufacturer's instructions. In brief, single-cell GEM (Gel Beads-in-Emulsion) were generated by the Chromium Controller, followed by GEM-RT (reverse transcription), Dynabeads cleanup, cDNA amplification, SPRIselect beads cleanup and Library construction. Quality, purity, size and concentrations of cDNA and libraries were assured by measurement on a Tapestation 2200 (Agilent Technologies). Indexed libraries were sequenced separately by either the Illumina NextSeq 500 platform or the NextSeq2000 sequencing platform (please refer to "instrument model" in the sample description section). Fixed snRNAseq: Protocol for probe hybridization, GEM generation and barcoding, amplification and library construction as per 10x Genomics Fixed RNA seq protocol CG000527_RevE, for multiplexed library (16 rxns x 16 BC): 1Mio cells per sample taken for probe hibridization; strategy washing -> pooling for pool preparation; targeted 80.000 cells per library. For scRNAseq of organoids: scRNA-seq with Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. Fixed snRNAseq: Chromium Fixed RNA Profiling for Multiplexed Samples.