 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 02, 2020 |
| Title |
Wheat_CS_10-day_shoots_ChIP_RNAPII |
| Sample type |
SRA |
| |
|
| Source name |
Seedlings
|
| Organism |
Triticum aestivum |
| Characteristics |
genotype: Chinese Spring
developmental stage: 14 days old seedlings
tissue: shoot
|
| Growth protocol |
Seeds were surface sterilized with bayrochlore and ethanol, sown on MS agar plates, placed at 4°C in the dark for 2 days and then grown in a growth chamber for 14 days (long day conditions, 18°C).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After fixation with 1% (v/v) formaldehyde, tissues were homogenized, nuclei isolated and lysed. Cross-linked chromatin was sonicated using Covaris S220. Protein/DNA complexes were immunoprecipitated with anti-RNA polymerase II CTD repeat YSPTSPS antibody (abcam ab26721) overnight at 4°C with gentle shaking and incubated for 1h at 4°C with 50 uL of Dynabeads Protein A (Invitrogen, Ref. 100-02D). The beads were washed 2 × 5 min in ChIP Wash Buffer 1 (0.1% SDS, 1% Triton X-100, 20mMTris-HCl pH 8, 2 mM EDTA pH 8, 150 mMNaCl), 2 × 5 min in ChIP Wash Buffer 2 (0.1% SDS, 1% Triton X-100, 20 mMTris-HCl pH 8, 2 mM EDTA pH 8, 500 mMNaCl), 2 × 5 min in ChIP Wash Buffer 3 (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 10 mMTris-HCl pH 8,1 mM EDTA pH 8) and twice in TE (10 mMTris-HCl pH 8, 1 mM EDTA pH 8). ChIPed DNA was eluted by two 15-min incubations at 65°C with 250?L Elution Buffer (1% SDS, 0.1 M NaHCO3). Chromatin was reverse-crosslinked by adding 20?L of NaCl 5M and incubating overnight at 65°C. Reverse-cross-linked DNA was treated with RNase and proteinase K, and extracted with phenol-chloroform. DNA was precipitated with ethanol in the presence of 20?g of glycogen and resuspended in 20?L of nuclease-free water (Ambion) in a DNA low-bind tube.
10 ng of IP or input DNA were used for ChIP-Seq library construction using NEB-Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's recommendations. For all libraries, ten cycles of PCR were used. The quality of the libraries was assessed with Agilent 2100 Bioanalyzer (Agilent), and the libraries were sequenced using 2x75bp pair-end reads on NextSeq 500 platform (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Adapters trimming: Sequencing reads were trimmed with trimmomatic with the following command "trimmomatic-0.36.jar PE -phred33 -validatePairs ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:5 TRAILING:5 MINLEN:20"
Read mapping: Trimmed reads data were mapped using bowtie2 v 2.3.3 with the following setting "bowtie2 --very-sensitive" against the genome of Triticum aestivum (iwgsc_1.0).
Filtering step: Mapped reads were sorted with sambamba view v.0.6.6 with the command "sambamba view -F "mapping_quality >= 10"
Duplicate filtering: duplicated reads were removed with sambamba markdup v. 0.6.6 with the command "sambamba markdup --io-buffer-size=8000 --overflow-list-size=1000000 -r"
Peak calling: peaks of read density were called with macs2 2.1.1.20160309 with the command "macs2 callpeak -f BAMPE --nomodel -q 0.001 --broad-cutoff 0.01 -g 17e9 --bw 300"
Bigwig generation: read density was scored over 100-bp non-overlapping bins with deepTools v.3.1.0 with the command "bamCoverage -p 24 -bs 100 -of bigwig --extendReads"
Genome_build: IWGSC_v1.0
Supplementary_files_format_and_content: bigwig file, containing Chr, start position, end position.
Supplementary_files_format_and_content: narrowPeak file from macs2
|
| |
|
| Submission date |
Jul 05, 2019 |
| Last update date |
Mar 02, 2020 |
| Contact name |
Lorenzo Concia |
| E-mail(s) |
colore@gmail.com, concia@bio.ens.psl.eu
|
| Phone |
769929568
|
| Organization name |
École Normale Supérieure
|
| Department |
Institut de Biologie
|
| Lab |
Plant and Algal Genomics Lab
|
| Street address |
46 Rue d'Ulm
|
| City |
Paris |
| ZIP/Postal code |
75020 |
| Country |
France |
| |
|
| Platform ID |
GPL26889 |
| Series (2) |
| GSE133880 |
Chromatin architecture in hexaploid wheat is hierarchically organized around genome territories and transcription factories (Wheat_ChIP) |
| GSE133885 |
Chromatin architecture in hexaploid wheat is hierarchically organized around genome territories and transcription factories |
|
| Relations |
| BioSample |
SAMN12219944 |
| SRA |
SRX6408586 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3929161_Wheat_Seedlings_ChIP_RNAPII.bigwig |
695.8 Mb |
(ftp)(http) |
BIGWIG |
| GSM3929161_Wheat_Seedlings_ChIP_RNAPII.q10.BAMPE.qval0.001.broad_0.01.macs2_peaks.narrowPeak.gz |
18.8 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
