title: GSM8673682: T47D cells, DMSO, total RNA, biol rep 3; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted from hypoxia and normoxia-treated T47D and H9 cells using TRIZOL. The quantity and quality of total RNA from hypoxia-treated T47D and H9 cells was assessed by Qubit (Life Technologies) and Bioanalyzer (Agilent Technologies), and 75 ng of RNA was used as input material to prepare nanoCAGE libraries as described previously (Lorent et al., EMBO, 2019) (T47D n = 3, H9 n = 4). Note that the Template Switching Oligonucleotides (TSO) include 8 nt random unique molecular identifiers (UMIs) that allow for the identification of duplicate transcripts arising from PCR amplification, and one of six barcodes for increased multiplexing (ACAGAT, GTATGA, ATCGTG, GAGTGA, TATAGC, GCTGCA). Sequencing was performed at the SciLifeLab NGI (Sweden) facility using the HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 100-base pair single-end setup using HiSeq Rapid SBS Kit vs chemistry.