title: GSM8889041: IMR32, ASO1, RNA-seq, rep4; Homo sapiens; RNA-Seq
library_construction_protocol: Attached cells were washes once with PBS, trypsinized, and collected by centrifugation. After two more PBS washes, pellets were redissolved in 700 uL QIAzol, incubated for 15 min at room temperature, snap-frozen, and stored at -80°C until further processing. Samples were unthawed at room temperature and 140 uL 100% chloroform was added. After vigorous mixing and centrifugation at 12 000 x g for 15 min at 4°C, the upper aqueous phase was collected to which 600 uL 100% EtOH was added. RNA from the mixture was isolated with the miRNeasy Micro kit following the manufacturer's instructions including a Dnase I digestion to remove contaminating DNA. RNA concentrations were measured by nanodrop. RNA was stored at -80°C. cDNA synthesis was performed with the iScript Advanced cDNA synthesis Kit according to the manufacturer's instructions with maximal shared RNA input across samples up to a maximum of 500 ng RNA template. cDNA was stored at -20°C. Total RNA libraries were prepared from 8 uL purified RNA using the SMARTer Stranded Total RNA-seq Kit v3 – Pico Input Mammalian following the manufacturer's instructions or TruSeq Stranded mRNA Library Prep Kit LT. RNA was fragmented at 94°C for 4 min prior to first strand cDNA synthesis. Library amplification was performed with 12 PCR cycles. Library size analysis was performed by running the libraries on a fragment analyzer. Library quantification was performed by RT-qPCR with the KAPA Library Quantification kit and libraries were pooled equimolarly. The final pool was quantified by Qubit. 675 pM of PicoV3-prepped libraries was loaded on a NextSeq 2000 instrument (NextSeq 2000 P2 v3, 2X 50 cycles, paired-end sequencing) with 2% PhiX or 2 pM of TruSeq-prepped libraries was loaded on a NextSeq 500 instrument (NextSeq 500/550 High-Output kit, 75 cycles, single-end sequencing) with 1% PhiX.