GSM5658217: MDA-MB-231 cells, biological replicate 1; Homo sapiens; OTHER
Extraction of nascent RNA was performed as described in Core et al 2008 and Wang et al 2011, with some adaptations: For each sample, nuclei from 1x107 MDA-MB-231 cells was used. All steps were done on ice. Cells were harvested by scraping and then resuspended in swelling buffer (10mM Tris-HCl pH 7.5, 2mM MgCl2, 3mM CaCl2) for 5 minutes on ice, then spun at 500 x g 5 minutes at 4°C. Swelling buffer was aspirated, and 10mL lysis buffer (10mM Tris-HCl pH 7.5, 2mM MgCl2, 3mM CaCl2, 0.5% IGEPAL CA-630, 10% glycerol) was added. The nuclei were then pelleted at 1000 x g for 5 minutes at 4°C. Lysis buffer was aspirated and nuclei resuspended in 1mL freezing buffer (50mM Tris-HCl pH 8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). Nuclei were then pelleted at 1000 x g 5 minutes 4°C. Freezing buffer was aspirated and nuclei resuspended in 100ul of freezing buffer and stored at -80°C. For the run-on assay, the frozen nuclei were thawed on ice and mixed with 100ul reaction buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 0.2units/ul SuperaseIN, 1% sarkosyl, 500uM each ATP, GTP, and Br-UTP, 2uM CTP), then incubated at 30°C for 5 minutes. Reaction was stopped by adding 600ul Trizol LS (Invitrogen), and RNA was isolated per the manufacturer's protocol. Libraries were constructed by first fractionating the RNA (in 20ul H2O) by adding 5ul 1M NaOH and incubating on ice for 40 minutes. Reaction was stopped by adding 25ul 1M Tris-HCl pH 6.8. This solution was run through micro biospin P-30 columns (BioRad), then DNase-treated by adding 10ul RQ1 DNase (Promega), 6.7ul 10X DNase reaction buffer, 1ul SuperaseIN and incubating at 37°C 10 minutes. This was run through a micro biospin P-30 column (BioRad). The RNA was dephosphorylated by adding 5ul Antarctic phosphatase (NEB), 8.5ul 10X phosphatase buffer, 1ul superaseIN, and incubated at 37°C for 1 hour. Labeled RNA was isolated with anti-BrdU agarose beads (Santa Cruz Biotech) that were prepared by incubating in blocking buffer (0.5X SSPE, 1mM EDTA, 0.05% tween-20, 0.1% PVP, 1mg/mL BSA) for 1 hour at 4°C, then resuspended in 500ul binding buffer (0.5X SSPE, 1mM EDTA, 0.05% tween-20). The RNA was added to the prepared beads after the RNA was heated at 65°C for 5 minutes, then placed on ice. The RNA was incubated with the beads with end-over-end rotation for 1 hour at 4°C. The beads were then washed 1X with low salt buffer (0.2X SSPE, 1mM EDTA, 0.05% tween-20), 2X with high salt buffer (0.5X SSPE, 1mM EDTA, 0.05% tween-20, 150mM NaCl), and 2X TET buffer (TE pH 7.4, 0.05% tween-20). RNA was eluted by adding 125ul elution buffer (5mM Tris-HCl pH 7.5, 300mM NaCl, 1mM EDTA, 0.1% SDS, 20mM DTT) and rotating end-over-end at room temp. Elution by repeated for a total of four times. RNA was isolated from the eluate by acid phenol chloroform extraction and precipitation. The RNA pellet was resuspended in 45ul H2O, and then phosphorylated by adding 5.2ul T4 PNK buffer, 1ul superaseIN, 1ul T4 PNK (NEB) and incubating at 37°C for 1 hour. RNA was isolated from the reaction by acid phenol chloroform extraction and precipitation. The RNA was polyA tailed by adding 0.8ul 10X polyA polymerase buffer, 1ul 1mM ATP, 0.5ul SuperaseIN, 0.75ul polyA polymerase (NEB), and incubating at 37°C for 30 minutes. RNA was then reverse transcribed by first adding 1ul 10mM dNTPs, 2.5ul 12.5uM oNTI223 primer, incubating at 75°C 3 minutes, then on ice 1 minute, then adding 2ul 10X superscript III buffer, 2ul 25mM MgCl2, 3ul 0.1M DTT, 0.5ul superaseIN, 1ul superscript III (Invitrogen), and incubating at 48°C for 20 minutes. Excess primer was digested by adding 4ul exonuclease I and incubating at 37°C for 1 hour. RNA was hydrolyzed by adding 1.8ul 1M NaOH and incubating at 98°C for 20 minutes, then adding 1.8ul 1M HCl to neutralize. The resulting cDNA was run on a 10% polyacrylamide TBEurea gel (Invitrogen) and the region from 110-405nt was excised and the DNA recovered by the crush soak method. The precipitated DNA was resuspended in 7.5ul H2O and circularized by adding 1ul CircLigase buffer, 0.5ul 1mM ATP, 0.5mM MnCl2, 0.5ul CircLigase (Lucigen) and incubated at 60°C for 1 hour, then 80°C for 20 minutes. Linearization was then performed by adding 3.8ul of 100mM KCl, 2mm DTT and 1.5ul APE1 (NEB), and then incubating at 37°C for 1 hour. The cDNA was run on a 10% polyacrylamide TBE-urea gel (Invitrogen) and the region from 125-305nt was excised and the DNA recovered by the crush soak method. The precipitated cDNA was resuspended in 20ul H2O. PCR was then performed by combining 10ul of the cDNA with 4ul 5X phusion HF buffer, 0.4ul 10mM dNTPs, 2ul each 5uM oNTI200 and oNT1201, 1.4ul H2O, 0.2ul phusion HF DNA polymerase (NEB) and running the following cycle: 98°C 30s, then repeat X13 98°C 10s, 60°C 15s, 72°C 15s. PCR product was run on an 8% polyacrylamide TBE gel, and the region from 150-230bp was excised. DNA was extracted by the crush soak method, and the library was sequenced.