Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
HEXIM1

Cell type

Cell type Class
Digestive tract
Cell type
HCT 116
Primary Tissue
Colon
Tissue Diagnosis
Carcinoma

Attributes by original data submitter

Sample

source_name
Hct 116 Cells
cell line
HCT116
cell type
Colonic Endothelial Cells
chip antibody
Hexim1 (Abcam; ab25388)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature in culture media, and the reaction was stopped by the addition of glycine (125 mM final). Cells were washed twice with PBS and re-suspended in 1 ml of nuclei extraction buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitor (Roche)) at 2x107 cells/ml and incubated for 10 min at 4°C. Cell nuclei were collected by centrifugation at 3000 rpm for 5 min at 4°C, and re-suspended in Szak's RIPA buffer (50 mM Tris-HCl pH 8.0, 1% NP-40, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 0.5 mM PMSF, protease inhibitor) at 5x107 nuclei/ml. Nuclear pellets were sonicated using the Bioruptor water bath sonicator (Diagenode) using the high setting with 30 sec on 30 sec off for 50 minutes to generate 100-300 bp DNA fragments. Approximately 25-50x106 nuclei were used for each ChIP. For the ChIP step, 2-4 ug of antibody was conjugated with 40 uL of protein G dynabeads (Invitrogen) and blocked with 0.16% bovine serum albumin for 1 hour at 4°C. Antibody-conjugated dynabeads were incubated with sheared chromatin suspension for 2 hr at 4°C. Then the beads were sequentially washed sequentially two times with the following buffers: Szak's RIPA buffer (see above), low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 20 mM Tris, pH 8.0), and 1 mM Tris-EDTA pH 8.0. Chromatin immunocomplexes were eluted by incubation for 10 min at 65°C with 1% SDS and 100 mM NaHCO3, and cross-linking was reversed by incubation in the solution adjusted to 200 mM NaCl and proteinase K (20 μg) for 1 hr at 65°C. DNA was then purified using the Zymo ChIP DNA Clean & Concentrator Kit (Zymo Research). Final ChIP DNA was quantified on a Qubit 2.0 Fluorometer (Invitrogen) and approximately 20 ng of DNA were submitted for library preparation. ChIP DNA was submitted to McDermott Center Sequencing Core at UT Southwestern Medical Center for library preparation and high-throughput sequencing. Samples were end repaired, 3’-end adenylated and barcoded with multiplex adapters (Applied Biosystems) and samples were PCR amplified (~14 cycles) and size selected with Ampure Beads. Sequencing was done using the Illumina NextSeq 500 using 50 bp read length.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
30612884
Reads aligned (%)
93.1
Duplicates removed (%)
20.2
Number of peaks
51766 (qval < 1E-05)

hg38

Number of total reads
30612884
Reads aligned (%)
93.9
Duplicates removed (%)
18.4
Number of peaks
51963 (qval < 1E-05)

Base call quality data from DBCLS SRA