title: GSM8262390: CHiPSC,Hypoxia,T21; Homo sapiens; RNA-Seq
library_construction_protocol: Cells from static culture, dissociated with TrypLE Select (Gibco Life Technologies) for 5 min at 37°C, and aggregates collected from the bioreactor were centrifuged at 300 x g for 5 min. These pellets were washed with DPBS, snap-freezed with liquid nitrogen and kept at -80°C. mRNA was extracted using the High Pure RNA Isolation kit (Roche) according to manufacturer's instructions. Roughly 100ng of total RNA were used for the transcriptomic interrogation of samples using Illumina's Stranded Total RNA Prep Ligation with Ribo-Zero Plus according to the manufacturer's instructions. Briefly, cytoplasmic and mitochondrial rRNAs as well as beta globin transcripts were depleted from the samples. The remaining RNA was fragmented and reverse-transcribed. A second strand cDNA synthesis step removed the RNA template while incorporating dUTP in place of dTTP in order to preserve strand specificity. Next, double-stranded cDNA was A-tailed, then ligated to Illumina anchors bearing T-overhangs. PCR-amplification of the library allowed the barcoding of the samples with 10bp dual indexes and the completion of Illumina sequences for cluster generation. Libraries were quantified with Qubit dsDNA HS Assay Kit and their profile was examined using Agilent's HS D1000 ScreenTape Assay. Sequencing was carried out in an Illumina NextSeq500 using single-end, dual-index sequencing (Rd1: 71 cycles; i7: 10 cycles; i5: 10 cycles) at a depth of 30 million reads per sample.