GSM7659967: 16190_1P_CB1_shTP53_Z05; Homo sapiens; RNA-Seq
RNA was isolated by separation of the aqueous phase with TRIzol Reagent (ThermoFisher, Bleiswijk, Netherlands) according to the manufacturer's protocol. The aqueous phase was mixed with 70% ethanol (1:1), and isolation was continued using the RNeasy microkit (Qiagen, Hilden, Germany) including on-column DNase I treatment. RNA libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) according to the manufacturer's protocol (KR1351–v1.16, Roche Sequencing Solutions). In brief, 25 ng to 1 µg of input RNA was depleted from ribosomal RNA by oligohybridization, RNaseH treatment, and DNase digestion. rRNA-depleted RNA was fragmented to ∼200-bp fragments, and first-strand synthesis was performed using random primers. The second strand was synthesized using dUTP for strand specificity. After adapter ligation, library amplification was performed, and the number of cycles was dependent on the amount of starting material. A bioanalyzer using a high-sensitivity DNA Chip (Agilent) was used to check fragment size. Samples were sequenced on an Illumina NextSeq 500 system with 2×43-bp paired-end sequencing (PE43)