Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
NEDD4

Cell type

Cell type Class
Breast
Cell type
MCF-7
Primary Tissue
Breast
Site of Extraction
Pleura
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
MCF7
cell type
human breast adenocarcinoma cell line
passages
8-13
cell line
MCF7
chip antibody
anti-NEDD4 (D17, sc-14482, Santa Cruz Biotechnology)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
34723251
Reads aligned (%)
85.4
Duplicates removed (%)
4.3
Number of peaks
1333 (qval < 1E-05)

hg38

Number of total reads
34723251
Reads aligned (%)
87.3
Duplicates removed (%)
2.8
Number of peaks
1103 (qval < 1E-05)

Base call quality data from DBCLS SRA