GSM8183362: Body (corpus), naive patient 9, scRNAseq; Homo sapiens; RNA-Seq
The biopsies were incubated with 0.5% DTT/HBSS w/o Ca & Mg [Roche #10197777001/Sartorius 02-018-1A] to reduce the mucus layer. Collagenase [Rosch #11213873001] at 1mg/ml was used for digestion in 20mM HEPES, 0.2% BSA in 1mM glucose DMEM [Roche #11213873001/Gibco 21885-025]. The biopsies were incubated twice for 15 min. at 37ºC. Crypts were dissociated to single cells with TrypLE/DNaseI [Gibco 12604-013/Roche 40276300] at 0.5mg/ml for 1-minute while pipetting, at room temperature. Cells were passed through a 30µm filter [MACS® smartstrainers]. Dead cells were removed using magnetic MicroBeads [Dead Cell Removal Kit Miltenyl #130-090-101] and then enriched for epithelial cells using magnetic MicroBeads [CD326 (EpCAM) MicroBeads Miltenyl #130-061-101]. Cells were separated into droplet emulsion using Chromium controller (10x genomics) and scRNAseq libraries were prepared and barcoded with UDIs using Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics), according to the manufacturer protocol. Sequencing was done using Illumina NextSeq 500 platform and Illumina NovaSeq 6000 platform with the following sequencing conditions: 28bp (Read1) and 54/56bp (Read2), and 28bp (Read1) and 90bp (Read2) respectevly.