Single-cell RNA-seq protocol for tissue biopsies

Biopsies were minced into small pieces with a scalpel and incubated at 37 °C for 5 minutes in freshly prepared dissociation buffer containing 2 mg/mL Collagenase P (Roche) and 0.2 mg/ml DNase I (Roche). Dissociated tissue was harvested and filtered through a 40 µm cell strainer (Flowmi Tipstrainers, VWR) into ice-cold PBS. Cells were collected by centrifugation at 300 g for 5 minutes at 4 °C before resuspension in Red Blood Cell lysis buffer (Merck) for 5 minutes, then centrifuged at 200 g for 5 minutes at 4 °C before resuspension in PBS containing 0.04% UltraPure BSA (AM2616, ThermoFisher Scientific), and finally strained through a 40-µm cell strainer to further remove cell clumps and large fragments. Cell number and viability was measured for the biopsy using Luna Cell counter as previously published^{63 (link)}. Libraries for scRNAseq were generated using the Chromium Single Cell 5′ library and Gel Bead & Multiplex Kit from 10x Genomics. We aimed to profile 5000 cells per library if sufficient cells were retained during dissociation. All libraries were sequenced on Illumina NextSeq until sufficient saturation was reached. After quality control, raw sequencing reads were aligned to the human reference genome GRCh38 and processed to a matrix representing the UMIs per cell barcode per gene using CellRanger (10x Genomics, v3.1).