Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
NR3C1

Cell type

Cell type Class
Lung
Cell type
A549
Primary Tissue
Lung
Tissue Diagnosis
Carcinoma

Attributes by original data submitter

Sample

source_name
GR_ChIP_exo
cell line
A549
cell treatment
100 nM dexamethasone 1 hr
chip antibody
GR Antibody E-20 (Santa Cruz Biotechnology, sc-1003)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For each replicate, 2 ×10^7 cells were fixed with 1% formaldehyde for 10 minutes. Fixation was quenced with 0.125 M glycine. Cells were washed with 1X PBS and collected and spun down in 5 mL of Farham Lysis Buffer (5 mM PIPES pH 8.0 / 85 mM KCl / 0.5% NP-40). Nuclei were re-suspended in 1 mL of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS at pH 7.4). Samples were sonicated using a Diagenode Bioruptor XL sonicator at 4°C to fragment chromatin to 200-500 bp segments. Insoluble components were removed by centrifugation for 15 min at 15000 rpm. We conjugated 5 µg of anti-GR (Santa Cruz Biotechnology, sc-1003) to 200 µl of sheep anti-rabbit IgG magnetic beads (Life Technologies 11203D/11201D). Sheared chromatin in RIPA was then added to the antibody-conjugated beads and incubated on a rotator overnight at 4°C. After incubation, beads were washed five times with a LiCl wash buffer (100 mM Tris at pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate), and remaining ions were removed with a single wash with 1 mL of TE (10 mM Tris-HCl at pH 7.5, 0.1 mM Na2EDTA) at 4°C. Chromatin-bound IgG beads were then washed with 10 mM Tris-HCl (pH 7.5) and resuspended in 30 µL of end repair mix: 1X T4 Ligase Buffer, 0.4 mM dNTPs, 1.8 U T4 DNA Polymerase, 6 U T4 PNK, and 3 U DNA Polymerase I, Large (Klenow) Fragment. Beads were incubated at room temperature for 30 minutes, and then washed with 10 mM Tris-HCl (pH 8). Beads were then resuspended in 30 µL of A-tailing mix: 1X NEB Buffer 2, 0.2 mM dATP, and 3 U Klenow Fragment (3'->5' exo-). Beads were incubated at 37℃ for 30 minutes. Next, beads were washed with 10 mM Tris-HCl (pH 7.5) and resuspended in 30 µL adapter ligation mix: 1X Quick Ligase Buffer, 4 µL Quick Ligase, and 1 pmol of Illumina Adapter A. Beads were incubated at room temperature for 30 minutes. Next, the beads were washed with 10 mM Tris-HCl (pH 9.5), and resuspended in 20 µL of λ-exonuclease mix: 1X λ-exonuclease Buffer and 5 U λ-exonuclease. The beads were incubated at 37℃ for 30 minutes. Next, the beads were washed with 10 mM Tris-HCl (pH 8), and resuspended in 20 µL RecJf mix: 1X NEB Buffer 2 and 15 U RecJf. The beads were incubated at 37℃ for 30 minutes, and then washed with TE Buffer. The beads were resuspended in 150 µL IP Elution Buffer (1% SDS, 0.1 M NaHCO3), and incubated for 1 hr at 65℃, with vortexing every 15 minutes. The beads were centrifuged at 20,000xg for 3 minutes, and the supernatant was transferred to a new tube and incubated at 65℃ overnight. DNA was purified using a MinElute PCR Purification kit (Qiagen), using 11 µL of EB Buffer for the elution. Purified DNA was mixed with 8 µL of phi29 DNA polymerase mix: 1X phi29 Buffer, 10 pmol primer P2, 375 nM dNTPs, and 4 ug BSA. The mix was heated to 95℃ for 2 minutes, 63℃ for 5 minutes, 30℃ for 2 minutes, and held at 30℃. 10 U of phi29 DNA polymerase was added to the mix, which was then heated at 30℃ for 20 minutes, 65℃ for 10 minutes, and held at 4℃. DNA fragments were purified using AxyPrep Mag PCR Clean-Up beads at a 2:1 bead volume:sample volume ratio. DNA was eluted off beads using 30 µL A-tailing mix, and incubated at 37℃ for 30 minutes. The DNA was then purified using AxyPrep Mag PCR Clean-Up beads as described above. DNA was eluted from beads using 30 µL of adapter ligation mix (with Illumina Adapter B instead of A), and incubated at room temperature for 30 minutes. The DNA was then purified using AxyPrep Mag PCR Clean-Up beads as described above, and eluted in 50 µL of PCR amplification mix: 1X Q5 Reaction Buffer, 200 nM dNTPs, 10 pmol Primer A, 10 pmol Primer B, and 1 U Q5 polymerase. PCR was carried out according to the manufacturer's specifications, using an annealing temperature of 65℃ , and an extension time of 30 seconds, for 20 cycles. The PCR products were purified with AxyPrep Mag PCR Clean-Up beads with a 1:1 ratio, and eluted in 30 µL of EB Buffer. DNA concentration was measured on a Qubit Fluorometer (Life Technologies), and fragment size distribution was assessed on a TapeStation 2200 (Agilent Technologies). Libraries were sequenced on an Illumina HiSeq 2000 with 50 base-pair paired end reads. Adapter A: (5'-CACTCTTTCCCTACACGACGCTCTTCCGATCT-3') + (5'-P-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT-3') Adapter B: (5'-ACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3') + (5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG-3') Primer P2: 5'-CAAGCAGAAGACGGCATACGAGAT-3’ Primer A: 5'-AATGATACGGCGACCACCGAGATCTACACTCT-3' Primer B: 5’-CAAGCAGAAGACGGCATACGAGATCGTGAT-3'

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
33730454
Reads aligned (%)
24.9
Duplicates removed (%)
86.0
Number of peaks
10584 (qval < 1E-05)

hg38

Number of total reads
33730454
Reads aligned (%)
26.1
Duplicates removed (%)
85.4
Number of peaks
10611 (qval < 1E-05)

Base call quality data from DBCLS SRA