GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM8063841 Query DataSets for GSM8063841
Status Public on Jun 03, 2024
Title Histone H3 MNase ChIP-seq low sedimentation rate (5-6) - Brd9-depleted auxin 20h replicate 2
Sample type SRA
Source name Brd9-3FTH-AID
Organism Mus musculus
Characteristics cell line: Brd9-3FTH-AID
cell type: embryonic stem cells
treatment: treated with auxin for 20 hours
strain: 129/Ola
chip antibody: histone H3 (Abcam ab1791)
Treatment protocol untreated
Growth protocol Mus musculus ES cells were grown on mitomycin C-inactivated Mus musculus embryonic fibroblasts at 37 °C, 5 % CO2, in DMEM (Sigma) supplemented by leukaemia inhibitory factor (LIF), 1X non-essential amino acids (Invitrogen), 15 % foetal calf serum (Invitrogen), 0.2 % β-mercaptoethanol (Sigma) and 1X penicillin/streptomycin (Invitrogen).
Extracted molecule genomic DNA
Extraction protocol Chromatin preparation: ES cells were collected and fixed in D15 culture medium containing 1 % formaldehyde (Sigma) for 10 min at 20°C. We stopped fixation by adding glycine to 125 mM, and the cells were centrifugated and washed three times with PBS. Cells were then permeabilized in Tris-HCl pH 7.5 15 mM, NaCl 15 mM, MgCl2 5 mM, EGTA 0.1 mM, KCl 60 mM, Sucrose 0.3 M, 0.4% IGEPAL CA-630 (I3021, Sigma) during 15 min on ice. We next diluted the cells with two volumes of MNase buffer (Tris-HCl pH 7.5 20 mM, NaCl 20 mM, CaCl2 2 mM, MgCl2 4 mM, KCl 15 mM, 0.1 M sucrose), and chromatin was digested during 10 min at 37 °C with 6 Kunitz units of MNase (New England Biolabs, 200 Kunitz units/µl) per 1 million cells. We stopped MNase digestion by adding a final concentration of 10 mM EDTA. We released chromatin from MNase-treated ES cells by passing the cell suspension 13 times through a 26G syringe. Next, we centrifuged the samples, and the supernatants containing the solubilized chromatin were used directly for ChIP-seq experiments or sucrose gradient centrifugation.
DNA libraries were prepared using MicroPlex Library Preparation Kit v2 or v3 (Diagenode)
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
Description ES cell chromatin was fragmented by micrococcal nuclease digestion and centrifugated for 16 hours through a 10-30 % sucrose gradient at 197,000 g, using a Beckman Coulter SW 41 swinging-bucket rotor. After centrifugation, 20 fractions were collected from top to bottom of the tube. MNase-ChIP-seq of low sedimentation rate fractions (pool of fractions 5 and 6) was performed.
Data processing Cleaning with fastp=0.20.1, parameters: --length_required 25 --detect_adapter_for_pe --cut_front --cut_tail --cut_window_size 6 --cut_mean_quality 10 --unqualified_percent_limit 40 --n_base_limit 7 --average_qual 0 –overrepresentation_analysis --trim_poly_g
Alignment with bowtie2=2.4.2, default parameters
Marking duplicates reads with picard=2.23.8, parameters: CREATE_INDEX=true VALIDATION_STRINGENCY=STRICT
Filtring with samtools=1.11, parameters: -h -f 1 -F 3852 -q 30
Coverage with deeptools=3.5.0 (for the blacklist :, parameters: bamCoverage --outFileFormat bigwig --binSize 1 --numberOfProcessors 5 --effectiveGenomeSize 2652783500 --normalizeUsing RPKM --MNase --minFragmentLength 10 --extendReads --centerReads --maxFragmentLength 500 --ignoreForNormalization chrX chrY chrM --blackListFileName mm9-blacklist.bed.gz
Assembly: mm9
Supplementary files format and content: bigWig
Submission date Feb 05, 2024
Last update date Jun 03, 2024
Contact name Matthieu Gerard
Organization name CEA
Street address Route Nationale
City Gif-sur-yvette
ZIP/Postal code 91191
Country France
Platform ID GPL9185
Series (1)
GSE255089 Nucleosomal and subnucleosomal organization of transcription cis-regulatory elements in mouse embryonic stem cells (chip-seq gradient)
BioSample SAMN39832145
SRA SRX23535732

Supplementary file Size Download File type/resource 190.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap