Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Epidermis
Cell type
Keratinocytes
MeSH Description
Epidermal cells which synthesize keratin and undergo characteristic changes as they move upward from the basal layers of the epidermis to the cornified (horny) layer of the skin. Successive stages of differentiation of the keratinocytes forming the epidermal layers are basal cell, spinous or prickle cell, and the granular cell.

Attributes by original data submitter

Sample

source_name
Human primary keratinocytes
cell type
Human primary keratinocytes engineered with stable IRF2 shRNA knockdown and a doxycyline inducible IRF2-3xHA allele
input
N/A
treatment
no treatment
barcode
TGCTGGGT
antibody
N/A
antibody maker
N/A
antibody catalog number
N/A

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitations were performed using the ChIPmentation protocol as described (Schmidl et al., 2015) with minor changes. The cells were trypsinized and 1 million cells were used for each replicate. Cells were fixed by adding formaldehyde to a final concentration of 0.8% in a crosslinking buffer (0.1M NaCl, 1mM EDTA, 0.5mM EGTA, 50mM Hepes, ph8.0) and incubated for 5 min on a rocker at 4°C. The fixation was terminated by incubating with 2.5 M Glycine for 5 min on a rocker at RT for 5 min. The cells were washed in ice cold PBS and resuspended in Hypotonic buffer (20mM Tris-HCl, 10mM NaCl, 3mM MgCl2) supplemented with Protease Inhibitors and 0.5mM PMSF and incubated on ice for 10 min. NP-40 (final 0.5%) was added to the mixture and vortexed for 20 sec. The nuclei were pelleted by centrifugation at 14000 rpm for 30 sec at 4°C. The pellet was resuspended in 1ml of 1X Wash Buffer C (truChIP Chromatin Shearing Kit) and incubated at 4°C for 10 min in a rocker. The nuclei were pelleted by centrifugation at 1700g for 5 min at 4°C and subsequently resuspended in shearing buffer D3 (truChIP Chromatin Shearing Kit) with Protease Inhibitors and 0.5mM PMSF, vortexed thrice and incubated at 4°C for 15 min in a rocker. Nuclear pellets were collected by centrifugation at 2000g for 5 mins at 4°C and resuspended in 130µl of TE (10mM Tris and 1mM EDTA) with 0.1% SDS and DNA was sheared at 4°C using a covaris LE220 sonicator (Covaris, Woburn, MA) for 4 minutes with 200 cycles per burst, 15% duty factor and a peak incident power of 300. Sonicated lysates were supplemented with salts and detergents to a final concentration of 1% Triton X-100, 150mM NaCl and 0.1% Na-deoxycholate. The chromatin was then cleared by centrifugation at 10,000g for 10 min and incubated with 5ul of Dynabeads™ Protein A (Thermo Fisher Scientific, Waltham, MA) for one hour to preclear the lysate. After collecting the input, the supernatant was incubated with end-over-end rotation overnight at 4°C with Dynabeads™ Protein A magnetic beads prebound with antibody. The antibody was bound to the beads by incubating the beads with the antibody in 200µl of bead binding buffer (TE, 0.2% Igepal) in a rotor at 4°C for 2 hours. Beads were washed once with low salt wash buffer (250mM NaCl, 10mM Tris-HCl, 1mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 0.1% Triton X-100), once with wash buffer containing 500mM NaCl, once with LiCl wash buffer (20mM Tris pH 8.0, 1mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), once with TE,0.1% Triton X-100 and twice with ice cold 10mM Tris-HCl. The beads and the input were then incubated at 37°C for 12 min with the Tagment DNA enzyme (Illumina, San Diego, CA), following tagmentation, the beads were washed twice with the low salt wash buffer and DNA was eluted TE Buffer, 250mM NaCl and 0.3% SDS. Cross-links were reversed by incubation first at 55°C and continued with protein digestion with the addition of Proteinase K for 10 hours at 64°C. DNA was purified using DNA Clean & Concentrator-5 (Zymo Research, Irvine, CA). Library preparations were performed as described with minor changes (Buenrostro et al., 2015). The amplified libraries were cleaned, and size selected using AMPure XP (Beckman Coulter, Indianapolis, IN). The libraries were sequenced using NextSeq® 500/550 High Output Kit v2 (75 cycles) (Illumina) in a NextSeq 550 (Illumina).

Sequencing Platform

instrument_model
NextSeq 550

hg38

Number of total reads
15301189
Reads aligned (%)
98.5
Duplicates removed (%)
5.6
Number of peaks
395 (qval < 1E-05)

hg19

Number of total reads
15301189
Reads aligned (%)
97.8
Duplicates removed (%)
6.6
Number of peaks
404 (qval < 1E-05)

Base call quality data from DBCLS SRA