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| Status |
Public on Aug 08, 2019 |
| Title |
input_BDR2_lib2 |
| Sample type |
SRA |
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| Source name |
seedlings, 8-day-old
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype: bdrs expressing Myc tag-BDR2
age: 8-day old
chip antibody: none
library prep kit: NEBNext Ultra DNA library Prep kit (E7370)
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| 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).
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| 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 DNA library prep kits (NEB, E7370 or E7645). Libraries were sequenced on a NextSeq 500 instrument at Indiana University Center for Genomics and Bioinformatics.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
library replicate with lower amount of starting DNA
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| 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.
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 : ChIP-seq peaks were detected using MACS2 (v. 2.1.0, Zhang et al., Genome Biology, 2008 ; 9 :R137) in paired-end mode using Myc_WT ( for BDR1 and BDR2) or input DNA (for FPA) 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 (FP10M : fragments per 10 million), i.e. the normalized number of fragments covering each base
Supplementary_files_format_and_content: bed files contain the location of peaks identified with MACS2
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| Submission date |
Apr 12, 2018 |
| Last update date |
Aug 08, 2019 |
| Contact name |
Pascal GP Martin |
| E-mail(s) |
pascal.martin@inrae.fr
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| Organization name |
INRAE
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| Department |
UMR1332 BFP
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| Lab |
FDFE
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| Street address |
71 avenue Edouard Bourlaux
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| City |
Villenave d'Ornon |
| ZIP/Postal code |
33140 |
| Country |
France |
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| Platform ID |
GPL19580 |
| Series (2) |
| GSE112443 |
RNA-seq, ChIP-seq and MNase-seq in WT, fpa mutant and bdr1,2,3 mutant Arabidopsis seedlings |
| GSE113059 |
Genome-wide occupancy of BDR1, BDR2 and FPA (ChIP-seq) |
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| Relations |
| BioSample |
SAMN08920118 |
| SRA |
SRX3928578 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3095572_GSF1369_B29_input_5ng_normcovFiltr.bigWig |
35.2 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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