GSM2155002: Gro-Seq plus E2; Homo sapiens; OTHER
10-20 millions of MCF7 cells treated with E2 or DEX for 1 hr were washed 3 times with cold PBS and then sequentially swelled in swelling  buffer (10mM Tris-HCl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5 min on ice, harvested, and lysed in lysis buffer (swelling buffer plus 0.5% NP-40, 20 units of SUPERase-In, and 10% glycerol). The resultant nuclei were washed two more times with 10ml lysis buffer. For the run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase-In, 1% sarkosyl, 500 mM ATP, GTP, and Br-UTP, 2 mM CTP) and incubated for 5 min at 30 C. The resultant nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent following manufacturer’s instructions. NRO-RNA was fragmented to 300-500nt by alkaline base hydrolysis on ice for 30 min and followed by treatment with DNase I and antarctic phosphatase. At this step, only a small portion of all the RNA species are BrU-labeled. To purify the Br-UTP labeled nascent RNA, the fragmented NRO-RNA was immunoprecipitated with anti-BrdU argarose beads in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween) for 1-3 hr at 4 C with rotation. Subsequently, T4 PNK was used to repair the ends of the immunoprecipitated Br-UTP labeled nascent RNA at 37 C for 1 hr. The RNA was extracted and precipitated using acidic phenol-chloroform. cDNA synthesis was performed as per a published method with few modifications. The RNA fragments were subjected to poly-A tailing reaction by poly-A polymerase for 30 min at 37 C. Subsequently, reverse transcription was performed using oNTI223 primer and superscript III RT kit. The cDNA products were separated on a 10% polyacrylamide TBE-urea gel and only those fragments migrating between 100-500bp were excised and recovered by gel extraction. Next, the first-strand cDNA was circularized by CircLigase and relinearized by APE1. Relinearized single strand cDNA (sscDNA) was separated on a 10% polyacrylamide TBE gel and the appropriately sized product (120-320bp) was excised and gel-extracted. Finally, sscDNA template was amplified by PCR using the Phusion High-Fidelity enzyme according to the manufacturer’s instructions. The oligonucleotide primers oNTI200 and oNTI201 were used to generate DNA for deep sequencing.  .
GEO Accession
GSM2155002