Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
CTCF

Cell type

Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
293T cells
cell type
HEK 293T cells
genotype
wide type
chip antibody
CTCF (Millipore, 07-729)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-nexus experiments were performed according to the published protocol50 with modifications. Briefly, HEK293T cells (~2x107) were crosslinked with 1% (v/v) formaldehyde for 10 min at room temperature (20-25°C), and then quenched by 125 mM glycine (final concentration). Crosslinked cells were lysed and washed twice using the ChIP buffer (10 mM Tris pH7.5, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS, 1x protease inhibitors, 0.15 M NaCl) to obtain nuclei. Nuclear samples were sonicated using Bioruptor Sonicator to fragmentize DNA to sizes ranging from 100 bp to 10,000 bp. Chromatins were then immunoprecipitated with 5-10 μl CTCF antibody (Millipore, 07-729) by slow rotation at 4°C for overnight. The mixture was then incubated with 50 μl Protein G Magnetic beads (life technologies, 10004D) for another 3 hours at 4°C. The magnetic beads with enriched chromatin were then sequentially washed with the washing buffer A (10 mM TE, 0.1% Triton X-100), washing buffer B (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 5.2% sucrose, 1.0% Triton X-100, 0.2% SDS), washing buffer C (250 mM NaCl, 5 mM Tris-HCl pH 8.0, 25 mM HEPES, 0.5% Triton X-100, 0.05% sodium deoxycholate, 0.5 mM EDTA), washing buffer D (250 mM LiCl, 0.5% NP-40, 10 mM Tris-HCl pH 8.0, 0.5% sodium deoxycholate, 10 mM EDTA), and the Tris buffer (10 mM Tris-HCl pH 7.5). The chromatin ends were repaired with 2 μl end-repair enzyme mixture (NEB, E6050S) in 50 μl 1x buffer for 30 min at 20°C and then washed again with the buffers A-D. The chromatin ends were dA-tailed by adding 3 μl Klenow exo- (NEB, M0212S) and 10 μl 1 mM dATP (NEB, N0440S) in 50 μl 1x NEB Buffer 2 at 37°C for 30 min. The Nexus adaptors (5' phosphate GATCG GAAGA GCACA CGTCT GGATC CACGA CGCTC TTCC, 5' phosphate TCAGA GTCGA GATCG GAAGA GCGTC GTGGA TCCAG ACGTG TGCTC TTCCG ATCT) were added by incubating with 50 μl 1x Ligase mix (NEB, M0367S) for 1h at 25°C. The ends were filled with 1 μl Klenow exo- and then with 1.5 μl T4 DNA polymerase (NEB, M0203S) to generate blunt-ended DNAs. Beads-DNA samples were then treated with 20 U Lambda Exonuclease (NEB, M0262S) with the Exonuclease buffer (0.2% Triton-X and 1% DMSO in 100 μl 1x Lambda) by incubation at 37°C for 1h with 1000 rpm on thermomixer. Samples were then digested by 75 U RecJf exonuclease (NEB, M0264S) with 0.2% Triton-X and 1% DMSO in 100 μl 1x NEB Buffer 2 at 37°C for 1h with 1000 rpm on thermomixer, and then washed three times with the RIPA buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40, 0.5 M LiCl). To elute DNA, incubate samples with 200 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) at 65°C for 30 min on thermomixer of 1000 rpm. The samples were reverse-crosslinked at 65°C for overnight, and then incubated with 2 μl RNase A (Thermo, EN0531) at 37°C for 2h, and then with 8μl proteinase K (NEB, P8107S) at 55°C for 4 h. The ssDNAs were extracted with 400 μl phenol:chloroform:isopentanol, and then precipitated with ethanol and sodium acetate. The precipitated DNAs were resuspended with 10 μl nuclease-free water. The ssDNA was incubated at 95°C for 5 min and then chill on ice immediately to denature single strand DNA. The ssDNA was circularized with 75 U ssDNA Ligase (Epicentre, CL4111K), 0.05 mM ATP, 2.5 mM MnCl2 in 15 μl 1x CircLigase Buffer at 60°C for 1h. The samples were incubated with 0.2 μM cut-oligo (GAAGA GCGTC GTGGA TCCAG ACGTG) in 50 μl 1x FastDigest Buffer (Thermo, FD0054) in thermocycler with the annealing program: 95°C for 1 min, slowly cool down at 1% ramp to 25°C for 1 min, hold at 25°C for 30min. Add 3 μl FastDigest BamHI and incubate at 37°C for 30 min to linearize DNA. Precipitate DNA using ethanol and sodium acetate, and resuspend DNA with 20 μl water. To PCR amplify samples, add 25 μl Q5 polymerase (NEB, M0543S), 2.5 μl 10 mM Illumina universal primer and index primer and follow the PCR program: 98°C for 30 sec to denature DNA, followed by 16 cycles of 98°C for 10 sec and 65°C for 75 sec, with a final extension at 65°C for 5 min. PCR products of sizes range about 150-300 bp were purified from 2% agarose gel using QIAquick gel extraction kit (Qiagen, 28604). Purified DNA samples were sequenced on an Illumina HiSeq platform.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
26750086
Reads aligned (%)
1.3
Duplicates removed (%)
10.4
Number of peaks
58 (qval < 1E-05)

hg38

Number of total reads
26750086
Reads aligned (%)
1.3
Duplicates removed (%)
10.3
Number of peaks
50 (qval < 1E-05)

Base call quality data from DBCLS SRA