title: GSM2157747: Input_siNedd4_EtOH; Homo sapiens; ChIP-Seq
library_construction_protocol: 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. 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.