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Sample GSM3819643 Query DataSets for GSM3819643
Status Public on Aug 08, 2019
Title Myc WT
Sample type SRA
Source name seedlings, 8-day-old
Organism Arabidopsis thaliana
Characteristics tissue: seedlings
ecotype: Col-0
genotype: wild-type
lineid: none
age: 8-day old
chip antibody: Anti-Myc tag, clone 4A6 (Millipore 05-724, lot 2824030)
library prep kit: NEBNext Ultra II DNA library Prep kit (E7645)
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 crosslinked for 15 min in 1 % formaldehyde under vacuum (5 min vacuum / vacuum release / 10 min vacuum) followed by 6 min quenching with glycine (125mM final concentration, under vacuum), extensive rinsing with ice-cold water and drying. Crosslinked 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) and filtered through 2 layers of Miracloth. Nuclei were prepared by 3 rounds of resuspension in extraction buffer (without spermine, twice with 0.25 % Triton X-100 and once with 0.1 % Triton X-100), incubation 12 min on ice and centrifugation at 1,000g for 12 min at 4°C (only 300g for the last centrifugation). The nuclei suspension was stored in 20 % glycerol at -70°C until used. After thawing on ice, nuclei were centrifuged for 10 min at 11,000g at 4°C and resuspended in nuclei lysis buffer (500 mM Tris-HCl, pH=8.0, 10mM EDTA, 1 % SDS, 1mM PMSF, 1 % plant protease inhibitor cocktail). Chromatin was sonicated for 12 min using a Covaris S220 (peak power=150, duty factor=20, cycles/burst=200). Soluble chromatin was recovered by centrifugation at 13,000g for 15 min at 4°C and diluted to (final concentrations): 20mM Tris-HCl, 150mM NaCl, 2mM EDTA, 0.1 % SDS, 1 % Triton X-100, 1mM PMSF, 1 % plant protease inhibitor cocktail.
Libraries were prepared using NEBNext Ultra II DNA library prep kits (NEB E7645). 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
Data processing Trimming: Trimmomatic (v0.38, Bolger, Lohse & Usabel, Bioinformatics, 2014 ; btu170) in paired end mode
Alignment: Bowtie2 (v, Langmead & Salzberg, Nature Methods, 2012 ; 9:357-59) with—very-sensitive and --dovetail option and a maximum insert size of 1Kb
Filtering: Duplicate reads were identified with Picard (v. 2.18.2) MarkDuplicates and removed. Samtools v 1.8 was used to keep only reads mapped in proper pairs with mapping quality (MapQ) above 2.
Coverage and normalization: Aligned reads were imported in R (v.3.4.3) to obtain coverages using Bioconductor (v3.6). Coverages were normalized as fragments per million (FPM) and exported to bigWig files with the rtracklayer package.
Peak calling: ChIP-seq peaks for BDR3 were detected from Myc_BDR3_rep2 using MACS2 (v. 2.1.1, Zhang et al., Genome Biology, 2008 ; 9:R137) in paired-end mode and Myc_WT as controls. peaks with a p-value<0.01 were kept. Peaks located in blacklisted regions were removed.
Genome_build: TAIR10
Supplementary_files_format_and_content: bigWig files contain the normalized coverage (FPM: fragments per million), i.e. the normalized number of fragments covering each base
Supplementary_files_format_and_content: bed files contain the location of BDR3 peaks or peak summits identified with MACS2
Submission date May 25, 2019
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
GSE131772 Genome-wide occupancy of BDR1 and BDR3 (ChIP-seq)
BioSample SAMN11858106
SRA SRX5900592

Supplementary file Size Download File type/resource
GSM3819643_normcovR_GSF2195-1_S19_filtered.bigWig 224.6 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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