high throughput sequence alignment protocol
Illumina FASTQ files were aligned to the human genome hg19 reference sequence (GRCh37) using TopHat v2.0.8b.
nucleic acid sequencing protocol
For sequencing libraries were diluted to 16 pM then applied to a V3 flowcell using the Illumina cBot according to manufacturer’s instructions. Sequencing was carried out on the Hi-Seq 2000 using HSCS v 188.8.131.52. Image analysis and basecalling were carried out using RTA 184.108.40.206. Deconvolution and fastq file production were performed with CASAVA 1.8.2.
HEK293 cells were transfected with Control or REST RNA interfeerence using lipofectmain for 24 hours. Cells were exposed to normoxic (21% oxygen) or hypoxic (1% oxygen) pre-conditioned media for 24 hours after transfection.
nucleic acid library construction protocol
For library construction the illumina TruSeq v2 mRNA kit was utilized after poly(A)+ RNA was enriched from total RNA samples. Libraries were visualized by Bioanalyzer (Agilent) using the High Sensitivity Chip. Libraries were quantified for pooling and sequencing using Kapa Biosystems qPCR quantitation kit.
HEK293 cells were cultured in DMEM containing 10% FCS and Pen/strep.
nucleic acid extraction protocol
RNA was isolated using QIAgen QIAquick columns.