Sample information curated by ChIP-Atlas


Antigen Class
TFs and others

Cell type

Cell type Class
Cell type
Primary Tissue
Site of Extraction
Tissue Diagnosis

Attributes by original data submitter


MCF7 cells, E2, 45m, p65 ChIP
cell line
cell type
breast cancer cell line
E2 for 45m
chip antibody
p65 (Vendor: Santa Cruz, cat# sc-372, lot# D0604)

Sequenced DNA Library

ChIP-Seq: Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adapter ligation as previously described (Heinz, S. et al. Molecular Cell 38, 576-589 (2010)) using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 12-15 cycles, size selected by gel extraction and sequenced on a HiSeq 2000 (Illumina) for 51 cycles. RNA-Seq: Enriched mRNA was hydrolyzed with Fragmentation Buffer (Ambion) for 10 min at 70°C and re-buffered to 10 mM Tris pH 7.4 using P-30 size exclusion columns (Bio-Rad). RNA was then de-capped for 2 h at 37°C with 0.5 µl (10 U/µl) TAP (Epicentre) in 20 µl TAP buffer containing 1 U/µl SUPERase-IN. Samples were 3’ dephosphorylated for 50’ at 37°C with 1 µl PNK (Enzymatics), 0.5 µl 10x TAP buffer, 1.5 µl water, 0.5 ul 0.25 M MgCl2 (3 mM free Mg2+ final), 0.5 µl 10 mM ATP (0.2 µM final to protect PNK). Subsequently, RNA was 5’-phosphorylated for 60 min at 37°C by adding 2 µl (10 U/µl) PNK, 10 ul 10x T4 DNA ligase buffer and 63 µl water. RNA was extracted with Trizol LS, precipitated in the presence of Glycoblue (Ambion), and dissolved in 4.5 µl water. 0.5 µl 9 µM of a 5’-adenylated sRNA3'MPX adapter /5Phos/AG ATC GGA AGA GCA CAC GTC TGA /3AmMO/ (IDT, desalted; adenylated with Mth ligase (NEB) according to the manufacturer’s instructions, phenol-chloroform/chloroform-extracted, ethanol-precipitated with glycogen and dissolved in water at 9 µM) were heat-denatured together with the RNA for 2 minutes at 70°C, and ligated with 100 U truncated T4RNA ligase 2 K227Q (NEB) in 10 µl 1x T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 hours at 16°C. To reduce adapter dimer formation, 0.5 µl 10 µM MPX_ RT primer 5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3’ (IDT, desalted) was added and annealed to the ligation product by incubating at 75°C for 2 minutes, then 37°C for 30 minutes, then 25°C for 15 minutes. Finally, 0.5 µl 5 µM hybrid DNA/RNA sRNA5'h adapter 5’-GTT CAG AGT TCT ACA rGrUrC rCrGrA rCrGrA rUrC-3’ (IDT) were ligated to previously capped RNA 5’ ends by adding 2 µl T4 RNA ligase buffer, 6 µl 50% PEG8000 (15% final), 1 µl 10 mM ATP, 9.5 µl water and 0.5 µl (5 U) T4 RNA ligase 1 for 90 minutes at 20°C. To 15 µl ligation reaction, an additional 0.5 µl 10 µM MPX_ RT primer were added, reactions were denatured at to 70°C for 1 minute, then placed on ice. RNA was reverse-transcribed by adding 3 µl 10x first strand buffer, 4.5 µl water, 1.5 µl 10 mM dNTP, 3 µl 0.1 M DTT, 1.5 µl RNaseOUT and 1 µl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. Complementary DNA was isolated by adding 35 µl AMPure XL beads (Beckman), binding and washing according to manufacturer’s instructions and dissolved in 40 µl TET. Libraries were PCR-amplified for 13 cycles with 0.75 µM primers oNTI201 (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAC G-3' and TruSeq-compatible indexed primers (e.g. 5’-CAA GCA GAA GAC GGC ATA CGA GAT iii iii GTG ACT GGA GTT CAG ACG TGT GCT CTT-3’ (desalted, IDT, i signifies index nucleotides) using Phusion Hot Start II (Thermo Scientific) in HF buffer containing 0.5 M betaine (98°C, 30s/12x(98°C, 10s/57°C, 25s/72°C, 20s)/ 72°C, 1min/4°C, ?), 175-225 bp fragments were size-selected on 10% PAGE gels and sequenced for 51 cycles on a HiSeq 2000 sequencer (Illumina) with small RNA sequencing primer 5’-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3’ and TruSeq Index sequencing primer (Illumina). Gro-Seq: Nuclei were extracted from 8-12 million cells grown on 10 cm plates and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments were purified on a denaturing 10% polyacrylamide TBE-urea gel. The recovered cDNA was circularized, linearized, amplified for 10-16 cycles. The final product was run on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. The final libraries were sequenced using an Illumina HiSeq 2000.

Sequencing Platform

Illumina HiSeq 2000


Number of total reads
Reads aligned (%)
Duplicates removed (%)
Number of peaks
270 (qval < 1E-05)


Number of total reads
Reads aligned (%)
Duplicates removed (%)
Number of peaks
209 (qval < 1E-05)

Base call quality data from DBCLS SRA