design: Effect of hypoxia (1% oxygen for 24 hours) on the expression and transcription of human HeLa, U87, MCF7 and HCT-116 cells
library_construction_protocol: RNA was extracted from both hypoxia-treated and control cells. A total of ~5-7.5*106 cells were used for total RNA extraction. HeLa, U87, MCF7, HCT-116 and D407 cells were grown in cell culture flasks (Thermo Fisher Scientific, Waltham, MA) in a Forma Steri-Cycle CO2 incubator (Thermo Fisher Scientific, Waltham, MA) in 37°C, 5% CO2. HeLa, U87 and MCF7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Biological Industries, Beit HaEmek, Israel) supplemented with 10% v/v fetal bovine serum (FBS, Biological Industries, Beit HaEmek, Israel), 2% v/v L-glutamine 29.2 mg/ml (SATORIUS, Göttingen, Germany), 1% v/v pen-strep (penicillin 10,000 units/ml, streptomycin 10 mg/ml, Biological Industries, Beit HaEmek, Israel) and 1% v/v MEM-Eagle non-essential amino acid solution (Biological Industries, Beit HaEmek, Israel). HCT-116 cells were grown in McCoy's 5A (Modified) Medium (manufactured by Thermo Fisher Scientific, imported by Rhenium, Modi'in-Macabim-Re'ut, Israel). Human D-407 retinal pigment epithelium cells, a kind gift from R.C. Hunt (57), were grown using DMEM medium supplemented with 3% FBS, 2mML-glutamine, 1,000 U/ml penicillin, and 1mg/ml strept Below 90% confluency, half of the cells were transferred to a CO2 incubator (Wiggens WCI-180, Straubenhardt, Germany), designed to maintain a humidified environment in 1% O2, 5% CO¬2 and 94% N2 for all experiments. All treated cells were incubated for 24 hours. Following exposure, the cells were immediately harvested and pooled for subsequent applications. To minimize oxygen exposure of the hypoxic cells during extraction, extra care was taken to keep the flask lids closed when removed from the incubator, followed by prompt cells harvesting.  RNA was extracted from both hypoxia-treated and control cells. A total of ~5-7.5*106 cells were used for total RNA extraction. Total RNA was extracted using Direct-zol™ RNA Miniprep kit (R2050, Zymo Research, Irvine, CA) with DNase I treatment. RNA integrity was determined on a QIAxcel device (QIAGEN, QIAxcel RNA QC Kit v2.0), and concentration was determined using a QuantiFluor® RNA System (Promega, #E3310) on a Qubit™ Flex Fluorometer (Invitrogen).  Stranded RNA-seq libraries were generated from 100ng of total RNA Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, #R3003). The molarity of libraries was determined using QIAxcel (QIAxcel DNA High Sensitivity Kit) and QuantiFluor® dsDNA System (Promega, # E2670) on a Qubit™ Flex Fluorometer (Invitrogen) Run-on transcription was performed as previously described(45) with minor modifications. Briefly, an aliquot containing 107 cells was incubated with 100 l of 2 x 1-Biotin run-on reaction mix: 10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM DTT, 150 mM KCl, 50 uM Biotin-11-CTP (Perkin Elmer), 50 M rCTP, 250 M rATP, 250 M rGTP, 250 M rUTP, 40 U SUPERase RNase Inhibitor (A&A Biotechnology) and 1% v/v Sarkosyl. The incubation was for 5 minutes at 37°C. Reactions were terminated by the addition of TRIzol LS (Thermo Fisher Scientific) and RNA was extracted according to manufacturer's protocol. Next, the RNA samples were heat denatured at 65°C for 40 seconds, placed on ice, and base-hydrolyzed for 8 minutes using ice-cold NaOH (0.2N final concentration). Hydrolysis was stopped using Tris-HCl pH 6.8 (0.5M final concentration) and the buffer was exchanged by running the samples through a P-30 column (Bio-Spin® columns with Bio-Gel®, Bio-Rad, Hercules, CA). The samples were enriched for Biotin-11-CTP-labeled RNA by a 20-minute incubation with magnetic streptavidin beads (Dynabeads M-280, Thermo-Fisher Scientific) followed by two washes with high salt buffer (50 mM Tris-HCl pH 7.4, 2M NaCl, 0.5% v/v Triton X-100), two washes with binding buffer (10 mM Tris-HCl pH 7.4, 300 mM NaCl, v/v 0.1% Triton X-100), and one wash with low salt buffer (5mM Tris-HCl pH 74, v/v 0.1% Triton X-100). Additionally, SUPERase RNase Inhibitor (New England Biolabs, NEB) was added to all buffers in a ratio of 2l/10ml. After a second TRIzol (Thermo Fisher Scientific) RNA extraction, the Rev3 RNA adapter (Table 1) was ligated to the 3' end of the RNA fragments by incubation with T4 RNA Ligase (NEB) at 20°C overnight (~16 hours). Samples were enriched for biotin-labeled RNA for a second time. Following the second enrichment cycle and RNA extraction, 5' ends of the precipitated RNA were repaired with RNA 5' pyrophosphohydrolase followed by phosphorylation with T4 polynucleotide kinase (both from NEB). Next, the 5' ends of the RNA molecules were ligated to the Rev5 RNA adapter (Table 1) by incubation overnight at 20oC. With both ends of the RNA ligated, a third enrichment cycle for biotin-labeled RNA was performed. The enriched RNA was subjected to a reverse transcription reaction with the RP1 reverse transcription primer (Table 2) and SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Next, the cDNA was PCR amplified using the Q5 High-Fidelity DNA Polymerase (NEB), along with the RP1 primer and a reverse primer (RPI-n) containing a unique, 6-nucleotide index sequence that differs between samples (Table 1). The amplified library was size selected using size exclusion polyacrylamide electrophoresis (200-1000 bp).