title: Illumina HiSeq 4000 paired end sequencing; Analysis of HIF dependent transcriptional regulation resolved by transcriptional start site, using 5′ end-Seq technology
design: Analysis of HIF dependent transcriptional regulation resolved by transcriptional start site, using 5′ end-Seq technology
library_construction_protocol: Two VHL-defective kidney cancer cell lines, RCC4 and 786-O, were used. Sublines of these cells with reintroduction of VHL or an empty vector control were prepared using lentiviral transduction, whereas HIF1B was inactivated using CRISPR/Cas9 mediated genome editing. Cells were grown in DMEM with 1 mM sodium pyruvate, 10% FBS and 5 µg/mL blasticidin at 37 ˚C in 5% CO2. For analysis of the effect of hypoxia, cells were cultured at 37 ˚C in 1% O2 and 5% CO2 for 24 hours.  An RNeasy Plus Mini Kit (QIAGEN, 74136) was used to extract RNA according to the manufacturer's instructions with the exception of technical replicate 2 of the samples from RCC4 cells. For these samples, cells were lysed with Buffer RLT Plus (QIAGEN, 1053393), and total RNA was extracted using an RNA clean and concentrator-25 kit (Zymo Research, R1018) according to the manufacturer's instructions, except that 752.5 µL of preconditioned RNA binding buffer (367.5 µL of RNA binding buffer (supplied with RNA clean and concentrator-25 kit), 367.5 µL of absolute EtOH and 17.5 µL of 20% SDS) was added to 350 µL of the cell lysate before loading onto the column. 5′ end-Seq was performed on the extracted total RNA. In short, a full-length cDNA library was generated using a combined reverse transcription and template switching reaction, followed by PCR amplification. The amplified libraries were multiplexed, and then purified using ProNex beads (Promega). The 3′ end of the cDNA library (i.e. 5′ end of mRNAs) was amplified specifically using Tn5 transposase-mediated tagmentation and PCR amplification with primers annealing to the appropriate adapters added by the template switching reaction and tagmentation. The PCR primers contained i5 and i7 Illumina index sequences and the amplified libraries were multiplexed.