title: GSM3555219: Groseq-plus E2 (14hrs) + E2 1hr + 2,5-HD; Homo sapiens; OTHER
library_construction_protocol: MCF7 cells after treatment were incubated in swellinig ice-cold buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5min. Cells were lysed in lysis buffer (swelling buffer with 0.5% IGEPAL and 10% glycerol). The resultant nuclei were washed once again with 10 ml lysis buffer and then re-suspended in 100μl of freezing buffer (50mM Tris-Cl pH 8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). GRO-seq experiments were performed as previously reported (Li et al., 2013). Briefly, MCF7 cells were swollen in swelling buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2) for 5 min on ice and then lysed in lysis buffer (swelling buffer with 0.5% IGEPAL and 10% glycerol), before being re-suspended in 100µl of freezing buffer (50mM Tris-Cl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For the run-on assay, re-suspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of Superase.In, 1% sarkosyl, 500µM ATP, GTP, and Br-UTP, 2µM CTP) and incubated for 5 min at 300C. The nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Invitogen) following manufacturer's instructions. After base hydrolysis on ice for 40min and followed by treatment with DNase I and antarctic phosphatase, the Br-UTP labeled NRO-RNA was purified by an anti-BrdU argarose beads (Santa Cruz Biotech) in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween) for 3hr at 4°C while rotating. Then T4 PNK (NEB) was used to repair the end of NRO-RNA. Subsequently, cDNA synthesis was performed as reported(9,33) with few modifications. The RNA fragments were subjected to poly-A tailing reaction by poly-A polymerase (NEB) for 30 min at 37°C. Reverse transcription was then performed using superscript III (Invitrogen) with oNTI223 primer. The cDNA products were separated on a 10% polyacrylamide TBE-urea gel with right product (~100-500bp) being excised and recovered by gel extraction. After that, the first-strand cDNA was circularized by CircLigase (Epicentre) and re-linearized by Ape1 (NEB). Re-linearized single strand cDNA were separated by TBE gel and the products of desired size was excised (~120-320bp) for gel extraction. Finally, cDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB) with primers oNTI200 and oNTI201 for deep sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3555219