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| Status |
Public on Feb 07, 2019 |
| Title |
ChIPseq_AGO4_DMS3-ZF_fwa_nrpd1_Figure6 |
| Sample type |
SRA |
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| Source name |
inflorescence
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: inflorescence
genotype: DMS3-ZF_in_fwa_x_nrpd1
ecotype: Columbia
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| Growth protocol |
All plants in this study were grown under long-day conditions (16h light/8h dark). Transgenic plants were obtained by agrobacterium-mediated floral dipping. T1 transgenic plants were selected on 1/2 MS medium + Glufosinate 50 μg/ml (Goldbio) or 1/2 MS medium + Hygromycin B 25 μg/ml (Invitrogen) in growth chambers under long day conditions and subsequently transferred to soil. Successive transgenic generations were germinated directly on soil.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIPs were performed as described previously with minor modifications (Johnson et al., 2014). 2g of inflorescences were used for Flag, HA, NRPE1 and AGO ChIPs. 2g of 12 day-old seedlings were used for Pol II Ser5 ChIP. All samples were ground and crosslinked with 1% formaldehyde. Chromatin was sheared using a Bioruptor Plus (Diagenode) and immunoprecipitated with Anti-Flag M2 (Sigma), Anti-HA 3F10 (Roche), Anti-Pol II phospho Ser5 Ab5131 (Abcam), Anti-NRPE1 and NRPE1 preimmune serum for NRPE1 control (Liu et al., 2018), Anti-AGO4, Anti-AGO6 and Anti-AGO9 (provided by Dr Olivier Voinnet, ETH, Switzerland) antibodies.
Libraries were prepared using the Ovation Ultra Low System V2 1-16 kit (NuGEN) following the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
ChIPseq for AGO4 in DMS3-ZF in fwa x nrpd1 used for Figure6
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| Data processing |
For ChIP-seq data analysis, raw reads were aligned to the Arabidopsis reference genome (TAIR10) with Bowtie (v1.0.0) (Langmead et al., 2009), allowing only uniquely mapping reads with fewer than two mismatches, and duplicated reads were removed with Samtools 0.1.19 (Li et al., 2009). For all ChIP-seq experiments, peaks were called using MACS2 (v 2.1.1.) (Zhang et al., 2008). To call peaks for DMS3-ZF (Flag) and ZF (HA) ChIP-seq, Flag and HA ChIP-seq in Col-0 was used as controls. Only Flag or HA peaks with more than 2 fold of enrichment were used for following analysis. DMS3-ZF sites without NRPE1 recruitment correspond to ZF off-target sites with greater than 4 fold enrichment and FDR of 0.05 (tested with R package DESeq (Anders and Huber, 2010) of Flag DMS3-ZF ChIP-seq over NRPE1 ChIP-seq normalized readcounts. The rest of the ZF off-targets were considered sites with NRPE1 recruitment. Genomic distribution of ChIP-seq peaks was calculated using HOMER (Heinz et al., 2010).
Genome_build: tair10
Supplementary_files_format_and_content: bigwig
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| Submission date |
Jan 07, 2019 |
| Last update date |
Feb 07, 2019 |
| Contact name |
Wanlu Liu |
| Organization name |
Zhejiang University
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| Department |
Zhejiang University - University of Edinburgh Institute
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| Lab |
Wanlu Liu
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| Street address |
718 East Haizhou Rd.,
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| City |
Haining |
| State/province |
Zhejiang |
| ZIP/Postal code |
314400 |
| Country |
China |
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| Platform ID |
GPL21785 |
| Series (2) |
| GSE124546 |
Co-targeting RNA Polymerases IV and V promotes efficient de novo DNA methylation in Arabidopsis |
| GSE124750 |
Co-targeting RNA Polymerases IV and V promotes efficient de novo DNA methylation in Arabidopsis [ChIP-seq] |
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| Relations |
| BioSample |
SAMN10701747 |
| SRA |
SRX5213637 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3553182_ChIPseq_AGO4_DMS3_ZF_fwa_nrpd1_Figure6.bw |
58.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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