title: GSM6862344: RNA-Seq_Eahy_WT_hypoxia_24h_clone7; Homo sapiens; RNA-Seq
library_construction_protocol: ChIP-seq libraries were prepared as previously described (Kaikkonen et al, Nucleic Acids Res., 2014, 42, 12570–84). Briefly, 1% formaldehyde fixed cells were collected, sonicated and H3K4me3 complexes were precipitated using rabbit polyclonal antisera to H3K4me3 (Abcam, #ab8580) with rabbit IgG used as a negative control. DNA was purified, blunted, and used for library preparation with ligation to NEXTflex DNA barcode adaptors and indexes (Bioo Scientific Corporation) were incorporated and amplified using PCR. Library fragments were size selected on 10% polyacrylamide gel and sequenced on a NextSeq 500 (Illumina) using single-end 75 cycles in high output mode. RNA was isolated using RNeasy minikit (Qiagen) and DNase treated with the RNase-free DNase set (Qiagen). RNA-Seq library preparation was carried out using a NEBNext Ultra II directional RNA library prep kit for Illumina (New England BioLabs) according to the manufacturer's instructions. The resultant library was quantified using a Qubit double-stranded-DNA HS assay kit (Thermo Fisher Scientific), and quality checked with a Bioanalyzer (Agilent Technologies). Individual libraries were pooled in equimolar amounts (4 nM for each) and sequenced with the NextSeq 550 sequencer (Illumina, San Diego, CA) using 75 single-end cycles for 10 million reads per sample.