GSM1613181: CD4+ T-cells; Homo sapiens; OTHER
Both primary and Jurkat CD4+ T-cells were maintained in RPMI-1640 media supplemented with 10% FBS, and treated for 30 minutes with low amounts of DMSO and ethanol (as they are controls for a separate experiment, manuscript in preparation).  To isolate nuclei, cells were resuspended in 1mL lysis buffer (10mM Tris-Cl pH 8, 300mM sucrose, 10mM NaCl, 2mM MgAc2, 3mM CaCl2, and 0.1% NP-40).  Nuclei were washed in 10mL of wash buffer (10mM Tris-Cl pH 8, 300mM sucrose, 10mM NaCl, and 2mM MgAc2) to dilute free NTPs.  Nuclei were washed in 1mL, and subsequently resuspended in 50uL, of storage buffer (50mL Tris-Cl pH 8.3, 40% glycerol, 5mM MgCl2, and 0.1mM EDTA), snap frozen in liquid nitrogen, and kept for up to 6 months before preforming PRO-seq. PRO-seq was run exactly as described in Kwak et. al. (2008) Science.  Briefly, nuclei were added to equal volume Nuclear Run-On(NRO) reaction mixture (10 mM Tris-HCl pH 8.0, 300 mM KCl, 1% Sarkosyl, 5 mM MgCl2, 1mM DTT, 500μM biotin-11-A/C/G/UTP (Perkin-Elmer), 0.8 u/μl RNase inhibitor) and incubated for 5 min at 30̊C. Nascent RNA was extracted using Trizol and precipitated in ethanol. Extracted nascent RNA was fragmented by base hydrolysis in 0.1 N NaOH on ice for 8-10 min, and neutralized by adding equal volume of 1 M Tris-HCl pH 6.8.  Excessive salt and residual NTPs were removed by using P-30 column (Bio-rad). Fragmented nascent RNA was bound to 30 μl of Streptavidin M-280 magnetic beads (Invitrogen) following the manufacturer’s instructions. The beads were washed once in high salt (2 M NaCl, 50 mM Tris-HCl pH 7.4, 0.5% Triton X-100), once in medium salt (300 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100), and once in low salt (5 mM Tris-HCl pH 7.4, 0.1% Triton X-100). Bound RNA was extracted from the bead using Trizol (Invitrogen) in two consecutive extractions.  RNA from each extraction was ethanol precipitated, re-suspended in depc H2O and pooled. Fragmented nascent RNA was re-suspended in depc H2O and ligated to a reverse 3' Illumina Hi-Seq adapter using T4 RNA ligase I (NEB) following the manufacturer's recommendations for 6 hours at 20C.  Ligated RNA was enriched with another round of purification on Streptavidin beads and Trizol extracted, as described above.  RNA products were treated with Tobacco Acid Pyrophosphatase (TAP, Epicentre) and Polynucleotide Kinase (PNK, NEB) to repair 5’ ends, and ligated to the reverse 5’ RNA adaptor for 6 hours at 20C.  The labeled RNA was purified a third time on Streptavidin beads.  Adaptor ligated nascent RNA was reverse transcribed using 25 pmol RT primer and amplified using Illumina TRU-seq barcodes.  Amplified product was PAGE purified and sequenced using Illumina HiSeq 2000 at Cornell University.
GEO Accession
GSM1613181