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| Status |
Public on Oct 27, 2022 |
| Title |
fwa_RNAseq_rep-3 |
| Sample type |
SRA |
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| Source name |
Arabidopsis floral tissue
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: floral tissue
developmental stage: 5-6 weeks
strain: Columbia-0
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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). Floral tissues were collected from 5-6 week plants
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| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP seq: Libraries were prepared with Ovation Ultra Low System V2 kits following the manufacturer’s instructions. BS seeq: 300 ng of DNA was sheared to 200bp with a Covaris S2 (Covaris). Libraries were prepared with the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer’s instructions. RNA seq: 1 ug of total RNA was used for library preparation with TruSeq Stranded mRNA kit (Illumina). Libraries were sequenced on HiSeq 2500 or NovaSeq 6000 (Illumina).
All Arabidopsis plants used in this study are in Columbia (Col-0) ecotype and were grown at 22°C in LD (16 hours light, 8 hours dark) conditions.
ATAC-seq: The nuclei collection process from inflorescence and meristem tissues is as described previously. Freshly isolated nuclei were used for ATAC-seq as described elsewhere. Inflorescence tissues were collected for extraction of nuclei as follows. About 5 g of inflorescence tissue 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 at 4°C 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. The ATAC-seq libraries were sequenced on HiSeq 2500 platform (Illumina).
Plants total RNAs were extracted with TRIzol and Direct-zol RNA Miniprep kit (Zymo, R2050). Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina) following the manufacturer instructions.
RNA-seq library preparation: Plants total RNAs were extracted with TRIzol and Direct-zol RNA Miniprep kit (Zymo, R2050). Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina) following the manufacturer instructions.
ChIP-seq library preparation: 10 grams of inflorescence and meristem tissues were used for Flag, HA, Pol II, H3, and H3K36me2/3 ChIP-seq. ChIP were performed as described previously60 with anti-FLAG M2 (Sigma), anti-HA (Roche), anti-Pol II (Ab26721, Abcam), anti-H3 (Ab1791, Abcam), anti-H3K36me2 (Ab9049, Abcam), and anti-H3K36me3 (Ab9050, Abcam) antibodies. Anti-NRPB1 antibody was raised in rabbits and further affinity-purified by ABClonal (China) using the peptide HEGDKKDKTGKKDASKDDK. To evaluate Pol II occupancy at both 5' end and 3' end of gene body, we selected anti-RNA Pol II CTD repeat YSPTSPS antibody which can capture Pol II signal at both regions (Ser5P and Ser2P). Libraries were prepared with NuGen Ovation Ultra Low System V2 kit following the manufacturer instructions.
Whole genome bisulfite sequencing (BS-seq) library preparation: Leaf tissue was used as starting material for BS-seq libraries preparation. Genomic DNA was extracted and converted with bisulfite treatment with EpiTect Bisulfite Kit (Qiagen) following the manufacturer instructions.
TSS-seq library preparation: TSS-seq was performed on 14 day old seedlings grown on MS plate following previous protocol. Heat stress was conducted by treating plants with 2 hours of 37°C. Five micrograms of total RNA were treated with DNase and CIP (NEB) to remove DNA and all non-capped RNA. Then 5’ caps of capped RNA were removed with Cap-Clip (CellScript) and single-stranded rP5_RND adapter were ligated to 5’-ends with T4 RNA ligase 1 (NEB). Ligated RNAs were enriched and captured by oligo(dT) Dynabeads (Thermo Fisher Scientific). Enriched samples were fragmented for 5 mins at 80°C and first-strand cDNA was generated with SuperScript III (Invitrogen) and random primers. Second-strand cDNA was synthesized with Phusion High-Fidelity DNA Polymerase (NEB) and the BioNotI-P5-PET oligo, and captured by Dynabeads for end repairing with End Repair Enzyme Mix (NEB) and ligation with barcoded Illumina compatible adapter using T4 DNA Ligase (NEB). TSS-seq sequencing libraries were amplified and size selected for single-end sequencing with NovaSeq 6000 platform (Illumina).
BS-PCR and McrBC assay for FWA tandem repeat: For BS-PCR of FWA tandem repeat, genomic DNA was extracted from leaf tissue of ASF1B-ZF T2 with CTAB-based method and converted using the EZ DNA Methylation-Lighting kit (ZYMO research). Methylation level of FWA promoter region and several control regions have been amplified with primers described previously46. HiSeq 2500 (Illumina) sequencing libraries were made from purified PCR products using a Kapa Hyper DNA Library Prep kit. For McrBC of FWA tandem repeat, genomic DNA was extracted from leaf tissue with CTAB-based method, and treated with PureLink RNase (Invitrogen) and then with McrBC (NEB) for 4 hours at 37°C. FWA tandem repeat region was quantified by qPCR.
ATAC-seq; RNA-seq; ChIP-seq; WGBS; TSS-seq; BS-PCR;
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RNA-seq data of fwa replicate 3
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| Data processing |
All libraries were sequenced at a length of 50 bps with HiSeq 2500 or NovaSeq 6000 platforms following manufacturer’s instructions (Illumina). Raw reads were aligned to the Arabidopsis reference genome (TAIR10) with Bowtie2 (v2.1.0)43, allowing only uniquely mapped reads with perfect matches. Duplicated reads were removed with Samtools (v1.9)44. Peaks were called using MACS2 (v2.1.1)45.
BS-seq reads were mapped to TAIR10 reference genome by bsmap (v2.90) with allowing 2 mismatches and 1 best hit (-v 2 -w 1)46. Reads with three or more consecutively methylated CHH sites were considered as non-converted reads and removed from the analyses. DNA methylation levels were calculated by #C/ (#C + #T). Differential Methylated Regions (DMRs) were called by methdiff function with every 100bp bin for where the difference in CG, CHG, and CHH methylation are at least 0.4, 0.2, and 0.1, respectively.
Genome_build: tair10
Supplementary_files_format_and_content: bed, bigwig, wig
ATAC-seq analysis: ATAC-seq reads adaptors were removed with trim_galore. The reads were then mapped to Arabidopsis thaliana reference genome TAIR10 using Bowtie2 (-X 2000 -m 1)62. Reads of chloroplast and mitochondrial DNA were filtered and duplicate reads were removed using Samtools 63. ATAC-Seq open chromatin peaks of each replicate were called using MACS2 with parameters -p 0.01 --nomodel --shift -100 --extsize 200. Consensus set of chromatin peaks of each samples were merged by bedtools (v2.26.0) intersect with allowing 10 base pairs distance64. edgeR was used to define significantly changed peaks [Fold Change, (FC) > 2 and False Discovery Rate, (FDR) < 0.05]65. Chromosomal distributions of ATAC-seq were calculated by dividing Arabidopsis chromosomes into 100 Kb sized bins and count reads at each bin with bedtools. ATAC-seq peak distribution was annotated with ChIPseeker66. Arabidopsis protein coding genes were ranked and divided into 10 quantiles according to gene coding region length, and coding region of each gene was divided in proportion into 10 quantiles. Transcriptional factor footprints were analyzed by TOBIAS67 with 572 plant TF motifs downloaded from Jasper (http://jaspar.genereg.net/).
RNA-seq analysis: Cleaned short reads were aligned to reference genome tair10 by Bowtie2 (v2.1.0), and expression abundance was calculated by RSEM with default parameters68. Heatmaps were visualized with the R package pheatmap. To reduce false positive of differential expression, transcripts with less than 5 reads of all replicates in total were regarded as lowly expressed genes and have been removed in subsequent analysis. Differential expression analysis was conducted using edgeR. A threshold of p value < 0.05 and Fold Change > 2 were used to decide whether significant expression difference exists between samples.
ChIP-seq analysis: ChIP-seq fastq reads were aligned to the TAIR10 reference genome with Bowtie (v1.1.2)70, allowing only uniquely mapping reads with 0 mismatches. Duplicated reads were removed by Samtools. ChIP-seq peaks were called by MACS2 (v2.1.1) and annotated with ChIPseeker71. Differential peaks were called by bdgdiff function in MACS2. ChIP-seq data metaplots were plotted by deeptools (v2.5.1)72. For Pol II 5’ occupancy analysis, Pol II occupancy was calculated based normalized reads count (RPKM) on a TSS +/- 200 bp region and a TSS +500 bp to TTS gene body region by bedtools.
Whole genome bisulfite sequencing (BS-seq) analysis: Trim_galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to trim adapters after filtering low quality reads. BS-seq reads were aligned to TAIR10 reference genome by Bismark (v0.18.2)73 with default settings. Reads with three or more consecutive CHH sites were considered as unconverted reads and filtered. DNA methylation levels were defined as #C/ (#C + #T). DMRs (Differentially Methylated Regions) were called by DMRcaller with p < 0.01 for where the differences in CG, CHG, and CHH methylation were at least 0.4, 0.2, and 0.1, respectively.
TSS-seq analysis: TSS-seq data were analyzed following previously published pipeline. TSS-seq reads were trimmed with Trim_galore and 5’ end UMI barcodes were trimmed using UMI-Tools. The reads were then aligned to TAIR10 genome assembly using STAR (v2.7.0e)76. Mapping files were filtered with MAPQ<10 and deduplicated using SAMtools (v1.9) before converted to stranded Bedgraph files using bedtools (v2.26.0). TSS peaks were identified with CAGEfightR (v0.99.0) and differential TSS peaks [Fold Change, (FC) >2 and p value<0.05] were called with DESeq2 (v1.28.1).
BS-PCR analysis: BS-PCR data were analyzed with a previously published pipeline. BS-PCR data were trimmed with primer sequences and mapped to TAIR10 reference genome with bsmap (v2.90) allowing 2 mismatches and 1 best hit (-v 2 -w 1).
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| Submission date |
Nov 09, 2021 |
| Last update date |
Oct 29, 2022 |
| 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) |
| GSE188493 |
Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis |
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| Relations |
| BioSample |
SAMN23010624 |
| SRA |
SRX13085612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5684038_fwa-rep3_count.txt.gz |
286.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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