GSM9056846: 44028_OB_11_3_BCGEtOH_A16; Homo sapiens; RNA-Seq
At day 6, cells were harvested by scraping, centrifuged, and resuspended in 350 μl of Lysis Buffer RA1 from the NucleoSpin RNA kit (Macherey-Nagel) and saved at -80°C until RNA isolation. RNA was isolated using the NucleoSpin RNA kit following the manufacturer's instructions. Total RNA was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems). 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 min. First strand synthesis, second strand synthesis and A-tailing was performed according to the protocol. For the adaptor ligation, a 1.5 µM stock was used (NextFlex DNA barcodes, Bio Scientific). First and second post-ligation cleanup was performed according to protocol. A total of 11 PCR cycles were performed for library amplification. The library amplification cleanup was done using a 0.8x followed by a 1.0x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer kit, and the library concentration was measured using the dsDNA High Sensitivity Assay (Denovix). Paired-end sequencing of reads of 50 bp were generated using an Illumina NextSeq 500.