Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
NR3C2

Cell type

Cell type Class
Unclassified
Cell type
Unclassified
NA
NA

Attributes by original data submitter

Sample

source_name
HK-GFP-MR cells_vehicle-treated
cell type
Human Kidney GFP-hMR cells
passage
18-26
chip antibody
Rabbit polyclonal 39N anti-MR antibody (custom-made)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Treated cells were fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Saint-Germain-en-Laye, France) for 10 min at RT. Fixation was quenched with 0.125 M glycine for 8 min. Cells were washed twice with ice-cold PBS. Cell lysis and chromatin shearing were carried out using the #HighCell ChIP kit buffers, according to manufacturer’s recommendations, with the following modifications; chromatin shearing was performed in 500 µL of shearing buffer S1 + Protease Inhibitor mix, applying 2 runs of 10 cycles at high intensity (Bioruptor®, Diagenode). Each cycle was composed of 30 s with effective application of ultrasounds (ON) and 30 s without (OFF). Between each run, heated water had to be removed and replaced by ice-cold water and ice. Concentrations of sheared chromatin protein were measured, using the bicinchoninic acid assay (Interchim, Montluçon, France) on a spectrophotometer Victor3® (Perkin Elmer, Courtabœuf, France), in parallel to a BSA standard range (Interchim). Protein A-coated magnetic beads (25 µl of the stock suspension) were washed three times in buffer C1 and re-suspended in C1 buffer supplemented with 1% PIC (100 µl final volume). Sheared chromatin (total amount of 1.3 mg protein) and 5 µg of one of the tested antibodies were then added to the beads and the final volume was adjusted to 500 µL with C1 buffer supplemented with 1% PIC and 0,1% BSA (final concentrations). Samples were incubated overnight at 4°C using a rotary incubator. Magnetic beads were then isolated and washed three times with C1 buffer and once with W1 buffer. DNA fragments from the immunoprecipitated chromatin were eluted with the DNA Isolating Buffer (DIB) supplemented with 1% Proteinase K, for 15 min at 55°C and finally for 15 min at 100°C. Reference for Antibody: Viengchareun S., et al, Osmotic stress regulates mineralocorticoid receptor expression in a novel aldosterone-sentitive cortical collecting duct cell line, Mol Endo, 2009, doi: 10.1210/me.2009-0095. In brief: Rabbit polyclonal anti-MR antibody (39N) was generated using the human MR 1 18 peptide and purified by affinity chromatography (Double X/XP boosting antibody production program, Eurogentec, Seraing, Belgium). Library preparation and Illumina sequencing were performed at the Ecole normale superieure Genomic Platform (Paris, France). Libraries were prepared using NEXTflex ChIP-Seq Kit (Bioo Scientific), using 1 ng of IP or Input DNA. Libraries were multiplexed by 6 on 1 flow cell lane. A 50 bp read sequencing was performed on a HiSeq 1500 device.

Sequencing Platform

instrument_model
Illumina HiSeq 1500

hg38

Number of total reads
37434880
Reads aligned (%)
96.5
Duplicates removed (%)
26.4
Number of peaks
918 (qval < 1E-05)

hg19

Number of total reads
37434880
Reads aligned (%)
95.6
Duplicates removed (%)
27.7
Number of peaks
1018 (qval < 1E-05)

Base call quality data from DBCLS SRA