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Sample GSM3095870 Query DataSets for GSM3095870
Status Public on Aug 08, 2019
Title H3K4me2_bdrs
Sample type SRA
Source name seedlings, 8-day-old
Organism Arabidopsis thaliana
Characteristics ecotype: Col-0
genotype: bdrs triple mutant
age: 8-day old
chip antibody: anti-H3K4me2 (Millipore 17-677, lot 2865656)
Growth protocol Seeds were sown on MS plates and stratified for 4 days at 4°C in the dark. Plates were transfered to a growth chamber at 21°C and grown for 8 days under long day conditions (16h light/8h dark).
Extracted molecule genomic DNA
Extraction protocol The seedlings were ground into fine powder in liquid nitrogen, incubated for 15 min at 4°C in extraction buffer (25mM Tris-HCl, pH=8.0, 0.44M sucrose, 10mM MgCl2, 0.1 % Triton X-100, 2mM spermine, 10mM beta-mercaptoethanol, 1 mM PMSF, 1 % plant protease inhibitor cocktail), filtered through 2 layers of Miracloth and centrifuged at 2000g for 13 min at 4°C. Nuclei were prepared by 2 rounds of resuspension in extraction buffer (without spermine, once with 0.2 % Triton X-100 and once with 0.1 % Triton X-100), incubation 6 min on ice and centrifugation at 1,000g for 12 min at 4°C. The nuclei suspension was stored in 20 % glycerol at -70°C until used. After thawing on ice, the nuclei were centrifuged for 10 min at 11,000g at 4°C and resuspended in MNase digestion buffer (10 mM Tris-HCl, pH=8.0, 1.5mM NaCl, 3mM CaCl2, 1mM MgCl2 , 1mM PMSF). Chromatin was digested with 400U MNase/mL (Thermo Fisher 88216) for 15min at 37°C. MNase digestion was stopped by adding EDTA/EGTA (25mM each final). Nuclei were centrifuged at 10,000g for 10 min at 4°C, the supernatant was kept and added to the soluble chromatin fraction following needle extraction. The pellet was resuspended in ChIP buffer (20mM Tris-HCl, pH=7.6, 50mM NaCl, 10mM EDTA, 0.1 % Triton X-100, 1mM PMSF, 1 % plant protease inhibitor cocktail) and soluble chromatin was recovered in ChIP buffer by 2 rounds of needle extraction (8 passage through 27.2G needle), incubation at 4°C for 20 min and centrifugation at 10,000g for 10 min.
Libraries were prepared using NEBNext Ultra II DNA library prep kits (NEB E7645) following the manufacturer's instructions. Libraries were sequenced on a NextSeq 500 instrument at Indiana University Center for Genomics and Bioinformatics.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description H3K4me2_optimal_peaks.regionPeak
Data processing Trimming : Trimmomatic (v0.33, Bolger, Lohse & Usabel, Bioinformatics, 2014 ; btu170) in paired end mode
Alignment : Bowtie2 (v 2.2.6, Langmead & Salzberg, Nature Methods, 2012 ; 9:357-59) with --dovetail option and a maximum insert size of 1Kb
Filtering : Duplicate reads were identified with Picard (v. 2.2.4) MarkDuplicates and removed. Samtools v 1.3 was used to keep only reads mapped in proper pairs with mapping quality (MapQ) above 2. Using samtools and awk (as in we further removed the reads corresponding to fragments of size below 70bp or above 250bp.
Coverage and normalization: Aligned reads were imported in R (v.3.3.2) to obtain coverages using Bioconductor (v3.4). Coverages were normalized as fragments per 10 million (FP10M) and exported to bigWig files with the rtracklayer package.
Peak calling: for each histone mark, peaks were called using MACS2 on pooled data from the 3 genotypes (WT, fpa, bdrs), using H3 ChIP-seq as control and following the Encode IDR pipeline (Landt et al., Genome Research, 2012 ; 22 :1813-1831). Irreproducible Discovery Rate (IDR) is described in Li et al., Ann Appl. Stat., 2011 ; 5(3):1752-79. The optimal set of peaks was defined using an IDR threshold of 0.01.
Genome_build: TAIR10
Supplementary_files_format_and_content: bigWig files contain the normalized coverage (FP10M : fragments per 10 million), i.e. the normalized number of fragments covering each base
Supplementary_files_format_and_content: regionPeak files contain the location of peaks detected using MACS2 and the IDR pipeline. Format is similar to the UCSC/Encode narrowPeak format ( Columns are : Chromosome, Start, End, Peak name, Score, SignalValue, Fold-change, log10(p-value), log10(q-value), summit location
Submission date Apr 12, 2018
Last update date Aug 08, 2019
Contact name Pascal GP Martin
Organization name INRAE
Department UMR1332 BFP
Street address 71 avenue Edouard Bourlaux
City Villenave d'Ornon
ZIP/Postal code 33140
Country France
Platform ID GPL19580
Series (2)
GSE112443 RNA-seq, ChIP-seq and MNase-seq in WT, fpa mutant and bdr1,2,3 mutant Arabidopsis seedlings
GSE113076 Genome-wide profiling of nucleosomes (MNase-seq), total H3, H3K4me2, H3K4me3 and H3K36me3 (native ChIP-seq) in wild-type, fpa mutant and bdrs triple mutant
BioSample SAMN08922497
SRA SRX3929422

Supplementary file Size Download File type/resource
GSM3095870_GSF1395_H3K4me2_tefs_Frag70to250.bigWig 93.2 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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