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| Status |
Public on May 28, 2016 |
| Title |
TWT_H3K27me3 |
| Sample type |
SRA |
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| Source name |
young seedlings
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| Organism |
Arabidopsis thaliana |
| Characteristics |
developmental stage: 10-day old whole seedlings
ecotype: C24
genotype: transgenic wild type
chip-antibodies: H3K27me3 (Abcam, ab6002, lot GR218433-4)
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| Treatment protocol |
No treatment
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| Growth protocol |
Seeds were grown on MS medium plates supplemented with 20 g/L sucrose and 0.8% agar at 22°C under long-day (23 h) illumination
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA-seq, total RNAs extracted from seedlings using the RNeasy Plant Mini Kit (QIAGEN). For ChIP-seq,A 2-g sample of the 10-day-old seedlings was crosslinked with 1% formaldehyde. Chromatin were isolated using nuclei extraction buffer (1 M hexylene glycol, 20 mM Tris-HCl (pH 8.0), 0.15 mM spermine, 5 mM 2-mercaptoethanol, 1% Triton X-100, 0.1 mM PMSF, and cocktail) and precipitated by centrifuging at 2,000 × g for 10 min at 4°C. After resuspension in 300 μL of nuclei lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% sodium dodecyl sulfate [SDS] and cocktail) at 4°C, the chromatin was sonicated into fragments of approximately 300 bp using a Bioruptor (15 cycles of 30 s on and 30 s off at high intensity)
For ChIP-seq library, a 10-ng quantity of ChIP or input DNA was used to prepare a high-throughput sequencing library. End repair, dA-tailing, adapter ligation, and amplification were carried out using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina (E6240, NEB) according to the manufacturer’s protocol. Index primers used for each sample were as follows: AGTCAA for TWT-H3K27me3; GTCCGC for TWT-H3K4me3; AGTTCC for TWT-H3K9me2; ATGTCA for TWT-H3; CCGTCC for TWT-Input; GTGGCC for fen-H3K27me3; GTGAAA for fen-H3K4me3; GTTTCG for fen-H3K9me2; CGTACG for fen-H3; and GAGTGG for fen-Input.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
For ChIP-seq analysis, reads were mapped to the Arabidopsis genome (TAIR10) using bowtie1 (version 0.12.9) allowing two nucleotide mismatches using following parameters: bowtie -q -k 1 -n 2 -l 36 --best -S -p 2. Mapped reads were de-duplicated and sorted by samtools (version 0.1.19-44428cd) Peaks were found using MACS software (version 1.4.2) using H3 as a background. For H3K27me3 peaks finding in the wild type as an example, the parameters were as follow: macs14 -t WT_K27ME3.sorted.bam –c TWT_H3.sorted.bam –f BAM -g 1.30e+8 -n WT_K27ME3 --shiftsize 73 --pvalue 1e-5 --bw=300 --mfold=10,30.TDF files were generated using igvtools count for IGV visualization.
For RNA-seq process, paired-end reads were selected and aligned to the Arabidopsis reference genome (version: TAIR10), using TopHat (v2.1.1) and Cuffdiff (v2.1.1). The output accepted_hits.bam files were used to generate TDF files by igvtools count for IGV visualization.
Genome_build: tair10
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| Submission date |
Mar 30, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jinkui Cheng |
| E-mail(s) |
chengjinkui@cau.edu.cn
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| Organization name |
China Agricultural University
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| Lab |
Zhizhong Gong
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| Street address |
No.2 Yuanmingyuan West Road
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| City |
Beijing |
| ZIP/Postal code |
100193 |
| Country |
China |
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| Platform ID |
GPL19580 |
| Series (1) |
| GSE79738 |
Requirement for flap endonuclease 1 (FEN1) to maintain genomic stability and transcriptional gene silencing in Arabidopsis |
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| Relations |
| BioSample |
SAMN04591550 |
| SRA |
SRX1670666 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2101793_TWT_H3K27me3.tdf |
19.5 Mb |
(ftp)(http) |
TDF |
| GSM2101793_TWT_K27ME3_peaks.bed.gz |
131.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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