 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 08, 2019 |
| Title |
RNAPII_WT_rep2_seq2 |
| Sample type |
SRA |
| |
|
| Source name |
seedlings, 8-day-old
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype: wild-type
age: 8-day old
chip antibody: anti-RNAPII (abcam ab817, lot GR299078-3)
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 7 or 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 DNA library prep kits (NEB, E7370 or E7645 as indicated). 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 |
sequencing replicate of RNAPII_WT_rep2_seq1
|
| 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.
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
|
| |
|
| Submission date |
Apr 12, 2018 |
| Last update date |
Aug 08, 2019 |
| Contact name |
Pascal GP Martin |
| E-mail(s) |
pascal.martin@inrae.fr
|
| Organization name |
INRAE
|
| Department |
UMR1332 BFP
|
| Lab |
FDFE
|
| 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 |
| GSE113078 |
Genome-wide profiling (ChIP-seq) of RNA polymerase II in wild-type, fpa mutant and bdrs triple mutant |
|
| Relations |
| BioSample |
SAMN08922556 |
| SRA |
SRX3929633 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3095918_GSF1413_P2_Col_1321_normcovFiltr.bigWig |
79.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
