GSM2157766: PRO_siCtrl_E2_Exp1; Homo sapiens; OTHER
GRO-seq experiments were performed as previously                     reported with a few modifications. Briefly, ~10 millions of MCF7 cells treated                     with E2 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 10 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 and finally resuspended in 100 µl of freezing                     buffer (50mM Tris-HCl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). 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, 100 µM A/GTP, 100 µM biotin-11-C/UTP (Perkin-Elmer)                     and incubated for 5 min at 30°C. The resultant nuclear-run-on RNA (NRO-RNA) was                     then extracted with TRIzol® LS reagent (Life Technologies, Cat# 10296-028)                     following manufacturer's instructions. NRO-RNA was fragmented to ~200-500nt by                     alkaline base hydrolysis on ice for 30 min and and neutralized by adding 1×                     volume of 1 M Tris-HCl pH 6.8, Excessive salt and residual NTPs were removed by                     using P-30 column (Bio-Rad, Cat# 732-6250), followed by treatment with DNase I                     (Promega Cat# M6101) and antarctic phosphatase (NEB Cat# M0289L). Fragmented                     nascent RNA was bound to 10 µl of MyOne Streptavidin C1 dynabeads (Invitrogen,                     Cat# 65001) following the manufacturer's instructions. The beads were washed                     twice in high salt (2 M NaCl, 50 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 0.5 mM                     EDTA), once in medium salt (1M NaCl, 5 mM Tris-HCl pH 7.5, 0.1% Triton X-100,                     0.5 mM EDTA), and once in low salt (5 mM Tris-HCl pH 7.5, 0.1% Triton X-100).                     Bound RNA was extracted from the bead using Trizol (Invitrogen, Cat# 15596-018)                     in two consecutive extractions, and the RNA fractions were pooled, followed by                     ethanol precipitation. The RNA fragments were then subjected to poly-A tailing                     reaction by poly-A polymerase (NEB, Cat# M0276L) for 30 min at 37°C.                     Subsequently, reverse transcription was performed using oNTI223 primer and                     superscript III RT kit (Life Technologies, Cat# 18080-044). The cDNA products                     were separated on a 10% polyacrylamide TBE-urea gel and only those fragments                     migrating between 100-400bp were excised and recovered by gel extraction. Next,                     the first-strand cDNA was circularized by CircLigase (Epicenter, Cat# CL4115K)                     and relinearized by APE1 (NEB, Cat# M0282L). Finally, cDNA template was                     amplified by PCR using the Phusion High-Fidelity enzyme (NEB, Cat# M0530L)                     according to the manufacturer's instructions. The oligonucleotide primers                     oNTI200 and oNTI201 were used to generate DNA library for deep sequencing and                     the primer sequences were described in previous paper. ChIP was performed as                     previously described. Briefly, cells were cross-linked with 1% formaldehyde at                     room temperature for 10 min. For KDM2A ChIP, cells were double cross-linked with                     2mM DSG (ProteoChem Cat# C1104) for 45 min and then for another 15 min with 1%                     formaldehyde (Sigma, F8775). In both situations, the cross-linking was quenched                     with 0.125M glycine for 5 min. Chromatin was fragmented using a Bioruptor                     (Diagenode) for 30 min at high power, with an interval of 30 s between pulses to                     get 200-500bp fragments and precleared using 20 µl Protein G Dynabeads (Life                     Technologies, Cat# 10009D). Subsequently, the soluble chromatin was incubated                     with 2-5 µg antibodies at 4°C overnight. Immunoprecipitated complexes were                     collected using 20 µl Protein G Dynabeads per reaction. We performed biotin ChIP                     or biotin ChIP-seq experiments for BLRP-tagged KDM2A and ERa, and ERa P-box                     mutation stable cell lines following an earlier protocol. Briefly, cross-linked                     protein-DNA complexes were pulled down by M-280 Streptavidin Magnetic beads                     (Life Technologies, Cat# 11205D) and the washing was performed under much more                     stringent conditions that included 2 washes with 1% SDS in TE (20 min each) and                     two washes with 1% Triton X-100 in TE. The washed streptavidin beads were then                     subjected to AcTEV protease (Life Technologies, Cat# 12575-015) digestion twice                     for tagged protein and DNA complex elution before de-crosslinking at 65°C                     overnight. For all ChIPs, after de-crosslinking overnight at 65°C, final ChIP                     DNA was extracted and purified using QIAquick spin columns (QIAGEN). The                     ChIP-seq libraries were constructed following Illumina's ChIP-seq Sample prep                     kit. The library was amplified by 14 cycles of PCR. GRO-seq experiments were                     performed as previously reported with a few modifications. Briefly, ~10 millions                     of MCF7 cells treated with E2 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 10 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 and finally                     resuspended in 100 µl of freezing buffer (50mM Tris-HCl pH8.3, 40% glycerol, 5mM                     MgCl2, 0.1mM EDTA). 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, 100 µM A/GTP, 100 µM biotin-11-C/UTP                     (Perkin-Elmer) and incubated for 5 min at 30°C. The resultant nuclear-run-on RNA                     (NRO-RNA) was then extracted with TRIzol® LS reagent (Life Technologies, Cat#                     10296-028) following manufacturer's instructions. NRO-RNA was fragmented to                     ~200-500nt by alkaline base hydrolysis on ice for 30 min and and neutralized by                     adding 1× volume of 1 M Tris-HCl pH 6.8, Excessive salt and residual NTPs were                     removed by using P-30 column (Bio-Rad, Cat# 732-6250), followed by treatment                     with DNase I (Promega Cat# M6101) and antarctic phosphatase (NEB Cat# M0289L).                     Fragmented nascent RNA was bound to 10 µl of MyOne Streptavidin C1 dynabeads                     (Invitrogen, Cat# 65001) following the manufacturer's instructions. The beads                     were washed twice in high salt (2 M NaCl, 50 mM Tris-HCl pH 7.5, 0.5% Triton                     X-100, 0.5 mM EDTA), once in medium salt (1M NaCl, 5 mM Tris-HCl pH 7.5, 0.1%                     Triton X-100, 0.5 mM EDTA), and once in low salt (5 mM Tris-HCl pH 7.5, 0.1%                     Triton X-100). Bound RNA was extracted from the bead using Trizol (Invitrogen,                     Cat# 15596-018) in two consecutive extractions, and the RNA fractions were                     pooled, followed by ethanol precipitation. The RNA fragments were then subjected                     to poly-A tailing reaction by poly-A polymerase (NEB, Cat# M0276L) for 30 min at                     37°C. Subsequently, reverse transcription was performed using oNTI223 primer and                     superscript III RT kit (Life Technologies, Cat# 18080-044). The cDNA products                     were separated on a 10% polyacrylamide TBE-urea gel and only those fragments                     migrating between 100-400bp were excised and recovered by gel extraction. Next,                     the first-strand cDNA was circularized by CircLigase (Epicenter, Cat# CL4115K)                     and relinearized by APE1 (NEB, Cat# M0282L). Finally, cDNA template was                     amplified by PCR using the Phusion High-Fidelity enzyme (NEB, Cat# M0530L)                     according to the manufacturer's instructions. The oligonucleotide primers                     oNTI200 and oNTI201 were used to generate DNA library for deep sequencing and                     the primer sequences were described in previous                     paper.