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| Status |
Public on Jun 12, 2015 |
| Title |
H3K4me3 |
| Sample type |
SRA |
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| Source name |
HK-GFP-MR cells_vehicle-treated
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Kidney GFP-hMR cells
passage: 18-26
chip antibody: anti-H3K4me3 (Diagenode, included in catalog numbers C01010050 AB-001-0010, C01010051 AB-001-0024)
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| Treatment protocol |
HK-GFP-MR cells, 7-8.10+6 by BP100, maintened in steroid-free medium for 24h, was stimulated by 100 nM of aldosterone or by the same volume of vehicle, for 1h.
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| Growth protocol |
Human Kidney GFP-hMR cells (HK-GFP-MR) were routinely cultured at 37°C, in an humidified incubator gassed with 5% CO2, seeded on petri dishes (BP100) with 10 mL of DMEM (DMEM High Glucose medium with L-glutamine) containing 2.5% fetal bovine serum (FBS) (Biowest, Courtaboeuf, France), 100X Penicillin Streptomycin. For the HK GFP-MR cells, the medium was supplemented with 200 µg/mL geneticin.
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
C11.8
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| Data processing |
Illumina bcl2fastq 1.8.4 software used for basecalling.
ChIP-Seq data analysis was done using the Eoulsan software version 1.2.2. Before mapping, poly N read tails were trimmed, reads ≤11 bases were removed, and reads with quality mean ≤12 were discarded. Reads were then aligned against the hg19 reference genome using Bowtie mapper (version 0.12.9) using the "-n 2 -l 28 -e 70 -k 2 --best" parameters. Only one alignment was kept in a given locus for each read and reads alignments matching on more than one locus were removed.
Peaks were called using macs 1.4.2 with the following setting: -gs hs -m 5,100, the other parameters remaining the default ones
Genome_build: hg19
Supplementary_files_format_and_content: bed format containg relative enrichments: vehicle vs input (C11.5 vs C11.1), aldosterone vs input (C11.6 vs C11.1), aldosterone vs vehicle (C11.6 vs C11.5) and H3K4me3 vs input (C11.8 vs C11.1).
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| Submission date |
Jun 11, 2015 |
| Last update date |
Jul 09, 2020 |
| Contact name |
Florian Le Billan |
| E-mail(s) |
florian.le.billan@utoronto.ca
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| Organization name |
INSERM
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| Department |
U1185
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| Street address |
63, Gabriel Péri street
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| City |
Le Kremlin-Bicêtre |
| ZIP/Postal code |
94276 |
| Country |
France |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE69777 |
Cistrome of the Aldosterone-activated Mineralocorticoid Receptor in Human Renal Cells |
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| Relations |
| BioSample |
SAMN03769807 |
| SRA |
SRX1056335 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1708663_C11.8_versus_C11.1.bed.bz2 |
235.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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