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| Status |
Public on Jun 09, 2023 |
| Title |
ChIP-seq data of wild type plants as control for PHD1-flag replicate 2 |
| Sample type |
SRA |
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| Source name |
floral tissue
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: floral tissue
cell line: Columbia-0
cell type: one month old
genotype: Col-0
treatment: anti-flag (M2 monoclonal, sigma F1804)
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| Growth protocol |
Arabidopsis plants of the Columbia-0 (Col-0) ecotype were used in this study. All plants were grown under green house condition (16 h light/8 h dark, 22C). For ChIP-seq and ATAC-seq, floral tissues were collected from about one-month-old plants. For BS-PCR and WGBS, rossette leaves were were collected from about one-month-old plants. For RNA-seq, twelve-day-old whole seedlings grown on half MS plates were collected.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA for WGBS was extracted with DNeasy Plant Mini Kit (QIAGEN). Genomic DNA for BS-PCR-seq was extracted with CTAB method. RNA was extracted with Direct-zol RNA MiniPrep kit (Zymo Research).
ChIP-seq library preparation: 15 ml of unopened flower buds were collected for each ChIP and flash-frozen in liquid nitrogen. The flower tissue was ground to fine powder with Retsch homogenizer in liquid nitrogen and resuspended in nuclei extraction buffer (50 mM HEPES pH 8.0, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 0.6% Triton X-100, 0.4 mM PMSF, 5 mM benzamidine, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), 50uM MG132). For transgenic lines of MOM1-MYC and PIAL2-MYC, EGS was first added to resuspended lysate to 1.5 mM and the tissue lysate was incubated at room temperature for 10 min with rotation. Then the lysate was supplemented with formaldehyde at 1% and rotated at room temperature for another 10 min followed by adding glycine to stop crosslinking. For ChIP of all other proteins, crosslinking was performed by directly supplementing formaldehyde to 1% without adding EGS, then rotated at room temperature for 10 min followed by adding glycine to stop crosslinking. The crosslinked nuclei were isolated, lysed with Nuclei Lysis Buffer and diluted with ChIP Dilution Buffer. Then the lysate was sonicated for 22 min (30 s on/30s off) with Bioruptor Plus (Diagenode). After centrifugation, antibody for FLAG epitope (M2 monoclonal antibody, Sigma F1804, 10 ul per ChIP) or for MYC epitope (Cell Signaling, 71D10, 20 ul per ChIP) were added to the supernatant and incubated at 4 ℃ overnight with rotation. Then, Protein A and Protein G Dynabeads (Invitrogen) were added and incubated at 4 ℃ for 2 hours with rotation. After that, the beads were washed and eluted, and the eluted chromatin was reverse-crosslinked by adding 20 ul 5 M NaCl and incubating at 65 ℃ overnight followed by treatment of Proteinase K (Invitrogen) for 4 hours at 45 ℃. DNA was purified and precipitated with 3 M Sodium Acetate, GlycoBlue (Invitrogen) and ethanol at -20 ℃ overnight. After centrifugation, the precipitated DNA was washed with ice cold 70% ethanol, air dried and dissolved in 120 ul of H2O. ChIP-seq libraries were prepared with Ovation Ultra Low System V2 kit (NuGEN).
RNA-seq library preparation: Twelve-day old seedlings grown on half MS medium (Murashige and Skoog Basal Medium) were collected and flash-frozen in liquid nitrogen. RNA was extracted with Direct-zol RNA MiniPrep kit (Zymo Research) and 1ug of total RNA was used to prepare RNA-seq libraries with TruSeq Stranded mRNA kit (Illumina)
WGBS library preparation: Rosette leaves of about one-month-old Arabidopsis plants were collected for DNA extraction using DNeasy Plant Mini Kit (QIAGEN). 500 ng DNA was sheared with Covaris S2 (Covaris) into around 200bp at 4℃. The DNA fragments were used to perform end repair reaction using the Kapa Hyper Prep kit (Roche), and together with Illumina TruSeq DNA sgl Index Set A/B (Illumina) to perform adapter ligation. The ligation products were purified with AMPure beads (Beckman Coulter), and then converted with EpiTect Bisulfite kit (QIAGEN). The converted ligation products were used as templates, together with the primers from the Kapa Hyper Prep kit (Roche) and MyTaq Master mix (Bioline) to perform PCR. The PCR products were purified with AMPure beads (Beckman Coulter) and used for sequencing.
ATAC-seq library preparation: About 5 grams of unopened flower buds was collected and immediately transferred into ice-cold grinding buffer (300 mM sucrose, 20 mM Tris pH 8, 5 mM MgCl2, 5 mM KCl, 0.2% Triton X-100, 5 mM β-mercaptoethanol, and 35% glycerol). The samples were ground with Omni International General Laboratory Homogenizer on ice and then filtered through a two-layer Miracloth and a 40-µm nylon mesh Cell Strainer (Fisher). Samples were spin filtered for 10 min at 3,000 g, the supernatant was discarded, and the pellet was resuspended with 25 ml of grinding buffer using a Dounce homogenizer. The wash step was performed twice in total, and nuclei were resuspended in 0.5 ml of freezing buffer (50 mM Tris pH 8, 5 mM MgCl2, 20% glycerol, and 5 mM β-mercaptoethanol). Nuclei were subjected to a transposition reaction with Tn5 (Illumina). For the transposition reaction, 25 µl of 2x DMF (66 mM Tris-acetate pH 7.8, 132 mM K-Acetate, 20 mM Mg-Acetate, and 32% DMF) was mixed with 2.5 µl Tn5 and 22.5 µl nuclei suspension at 37°C for 30 min. Transposed DNA fragments were purified with ChIP DNA Clean & Concentrator Kit (Zymo). Libraries were prepared with Phusion High-Fidelity DNA Polymerase (NEB) in a system containing 12.5 µl 2x Phusion, 1.25 µl 10 mM Ad1 primer, 1.25 µl 10 mM Ad2 primer, 4 µl ddH2O, and 6 µl purified transposed DNA fragments.
BS-PCR library preparation: Rosette leaves of about one-month-old plants were collected and subject to DNA extraction with CTAB method followed by bisulfite DNA conversion using the EpiTect Bisulfite kit (QIAGEN) kit. Three regions of the FWA gene were amplified from the converted DNA with Pfu Turbo Cx (Agilent): Region 1 (chr4: 13038143-13038272), Region 2 (chr4: 13038356- 13038499) and Region3 (chr4: 13038568-13038695). Libraries were prepared with the purified PCR product by the Kapa DNA Hyper Kit (Roche) together with TruSeq DNA UD indexes for Illumina (Illumina) and were sequenced on Illumina iSeq 100 or HiSeq 4000 instruments.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
For ChIP-seq analysis, raw reads were trimmed using trim_galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and aligned to the TAIR10 reference genome with bowtie2 (v2.4.2) allowing zero mismatch and reporting one valid alignment for each read. The Samtools (v1.15) were used to convert sam files to bam files, sort bam files and remove duplicate reads. Track files in bigWig format were generated using bamCoverage of deeptools (v3.5.1) with RPKM normalization. Peaks were called with MACS2 (v2.1.2) and peaks frequently identified in previous ChIP-seq of Col-0 plant with M2 antibody for FLAG epitope were removed from analysis.
For RNA-seq analysis, the raw reads were aligned to the TAIR10 reference genome with bowtie2. Rsem-calculate-expression from RSEM with default settings was used to calculate expression levels. DEGs and DE-TEs were calculated with run_DE_analysis.pl from Trinity version 2.8.5 and log2 FC ≥ 1 and FDR < 0.05 were used as the cut off. RNA-seq track files in bigWig format were generated using bamCoverage of deeptools (v3.1.3) with RPKM normalization.
For WGBS analysis, the raw reads were aligned to both strands of the TAIR10 reference genome using BSMAP (v.2.74), allowing up to 2 mismatches and 1 best hit. Reads with more than 3 consecutives methylated CHH sites were removed, and the methylation level was calculated with the ratio of C/(C+T).
For BSPCR analysis, raw reads were aligned to both strands of the TAIR10 reference genome with BSMAP (v.2.90) allowing up to 2 mismatches and 1 best hit. After quality filtering, the methylation level of cytosines was calculated as the ratio of C/(C+T).
For ATAC-seq analysis, raw reads were adaptor-trimmed with trim_galore and mapped to the TAIR10 reference genome with Bowtie2 (-X 2000 -m 1). After removing duplicate reads and reads mapped to chloroplast and mitochondrial, ATAC-Seq open chromatin peaks of each replicate were called using MACS2 with parameters -p 0.01 --nomodel --shift -100 --extsize 200. Consensus peaks between replicates were identified with bedtools (version 2.26.0) intersect and differential accessible peaks were called with the R packge edgeR (version 3.30.0).
Assembly: tair10
Supplementary files format and content: .bw files are bigWig files of coverage tracks normalized with RPKM (for ChIP-seq and ATAC-seq), or methylation levels (for WGBS)
Supplementary files format and content: _methratio_type.txt files are tab-delimited text files containing methylation type and level at indicated chromosome positions.
Supplementary files format and content: .genes.results files are tab-delimited text files containing RNA-seq quantification data over genes and TEs.
Supplementary files format and content: .narrowPeak files are bed files containing position of called peaks for ChIP-seq.
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| Submission date |
Dec 23, 2022 |
| Last update date |
Jun 09, 2023 |
| Contact name |
Zhenhui Zhong |
| E-mail(s) |
zhenhuizhong@gmail.com
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| Organization name |
University of California, Los Angeles
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| Department |
Department of Molecular, Cell and Developmental Biology
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| Lab |
Jacobsen Lab
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| Street address |
610 Charles E Young Dr East
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| City |
Los Angeles |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL21785 |
| Series (1) |
| GSE221679 |
The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation. |
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| Relations |
| BioSample |
SAMN32387881 |
| SRA |
SRX18840017 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6892956_ChIP_WT_control_forPHD1flagrep2.bw |
43.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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