GSM5777330: Micro-organoid-STAR-high_2; Homo sapiens; RNA-Seq
RNA was extracted using the RNeasy Mini RNA extraction kit (Qiagen, Cat. No. 74106) according to the manufacturer's instructions with DNaseI treatment. Up to 73ng RNA per sample was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (KAPA Biosystems). In short, oligo hybridisation 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 94⁰C for 6:30 min. First strand synthesis, second strand synthesis, and A-tailing were performed according to protocol. For the adapter ligation, a 1.5μM stock was used (NextFlex DNA barcodes, Bioo Scientific). First and second post-ligation cleanup were performed according protocol. The library was amplified in 13 cycles and the subsequent cleanup was performed using a 0.8x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer (Agilent Technologies) and the library concentration was measured using the KAPA Library Quantification Kit (KAPA Biosystems). Sequencing was performed using an Illumina NextSeq 500, while 50‐bp paired‐end reads were generated.