GSM9871229: Immunotherapy colitis 001; Homo sapiens; RNA-Seq
For bulk RNA sequencing, archival FFPE pinch biopsies were sectioned at 5 or 10 μm. RNA isolation was performed with RNeasy FFPE Kit according to manufacturer's instruction. RNA quality assessment was performed using Qubit (concentration) and TapeStation (RIN values) and 120 ng per sample was collected for cDNA conversion QUOTED from publication Wester et al iScience 2021 ( doi: 10.1016/j.isci.2021.103444):  A total amount of 1 μg RNA per mouse small intestinal sample or 100ng RNA per CRC organoid sample was used for the preparation of RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (KAPA Biosystems). In short, oligo hybridization and rRNA depletion, rRNA depletion cleanup, Dnase digestion, Dnase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94 C for 6:30 min. First strand synthesis, second strand synthesis, and A- tailing was performed according to protocol. For the adapter ligation, a 7 μM stock was used (NextFlex DNA barcodes, Bioo Scientific). First, and second post-ligation cleanup was performed according protocol. For the library amplification, 6 cycli were used. The library amplification cleanup was performed using a 0.8x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer (Agilent Technologies), library concentration was measured using the DeNovix dsDNA High Sensitivity Assay (DeNovix).