Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Lung
Cell type
WI-38
Primary Tissue
Lung
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
Human fetal lung fibroblats (Oncogene induces senescent)
antibody
input
cell type
WI-38hTERT/GFP-RAF1-ER
sorting
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed on plate with 1% formaldehyde solution (Pierce; 28908) in complete MEM fro 10 minutes. 125mM glycine (Final) was then added and insubated for 5 minutes to quench formaldehyde. The cells were then scraped and the cells were immediately pelleted, frozen in liquid nitrogen and stored at -80C until further use. 1x10^6 cells per IP for chromatin marks, or 2.5x10^6 cells for CTCF were then thawed on ice, resuspended in cold cell lysis buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40) + 1xEDTA-free Protease Inhibitors. Cells were lysed for 30min, and resuspended in 50ul per IP cold nuclei lysis buffer (50mM Tris pH8, 10mM EDTA, 1% SDS) + 1xProtease Inhibitors. Nuclei were lysed for 60min at 4C with rotation and then sonicated for 16-18 cycles using Bioruptor (Diagenode). After sonication, 10x volumes of IP dilution buffer (20mM Tris pH8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS + protease inhibitors) was added, chromatin was precleared using 25ul Protein A dynabeads (ThermoFisher, Cat.N: 10002D) / 1mL for 1hrs at 4C with rotation. Meanwhile, 25ul beads / IP were washed once with cold 0.5% BSA in PBS, and incubated with the antibody for 4-5hrs at 4C in 0.5ml 0.5% BSA in PBS). Beads were then washed once with 0.5% BSA in PBS, added to the precleared chromatin and incubated overnight at 4C with rotation. Beads were then washed once with cold IP wash buffer 1 (20mM Tris pH8, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt wash buffer (20mM Tris pH8, 2mM EDTA, 500mM NaCl, 1% Triton X-100,0.1% SDS), once with cold IP wash buffer 2 (10mM Tris pH8,1mM EDTA,250mM LiCl,1% NP-40,1% sodium deoxycholate) and twice with cold TE buffer (1mM Tris pH8, 1mM EDTA). DNA:protein complexes were then eluted twice for 15min at 65C in 100ul elution buffer (100mM NaHCO3, 1%SDS) each time. 16ul 5M NaCl was then added and samples + inputs were reverse cross-linked at 65C, RNase A and proteinase K treated and purified using ultrapure phenol/chloroform (ThermoFisher, Cat.N: 15593-049).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
32900516
Reads aligned (%)
98.7
Duplicates removed (%)
14.0
Number of peaks
675 (qval < 1E-05)

hg19

Number of total reads
32900516
Reads aligned (%)
97.6
Duplicates removed (%)
14.3
Number of peaks
358 (qval < 1E-05)

Base call quality data from DBCLS SRA