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| Status |
Public on Aug 11, 2021 |
| Title |
RNAseq of Arabidopsis: ddm1nrpd1_rep2 |
| Sample type |
SRA |
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| Source name |
seedling
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| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: ddm1nrpd1
age: two-week-seedling
tissue: seedling
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| Growth protocol |
Arabidopsis seeds were plated on 1/2 Murashige and Skoog (MS) medium with 0.6% agar and 1.5% sucrose and stratified for 7 days at 4℃ in darkness before being transferred to the growth chamber (16 h light/8 h dark, 22℃)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from two-week-old seedlings using the RNeasy Plant Mini kit (Takara)
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
For RNA-seq data, low-quality sequences and adaptors were trimmed using Trimmomatic. Clean reads were mapped to the reference genome by TopHat with the parameter "-g 1". The total number of reads mapping to each gene was calculated with the htseq-count script in HTSeq with the parameter “--nonunique all”, to minimize over-estimation of TE expression caused by overlapping genes; read counts for each TE were calculated by htseq-count with parameters "--nonunique all -m intersection-strict" based on the TE-only annotation. Genes and TEs with a normalized expression level of at least one count per million mapped reads (CPM) in three or more libraries were considered as expressed. Principle component analysis (PCA) based on the transcript levels of the expressed protein-coding genes and TEs was performed using the prcomp function in R software with default settings. Differentially expressed TEs with at least a 2-fold change in expression and an FDR < 0.05 were identified by the R package edgeR using the trimmed mean of M-values (TMM) method.
For methylation data, low-quality sequences and adapters were trimmed using Trimmomatic with parameters “LEADING: 3 SLIDINGWINDOW: 4: 30 MINLEN: 36”, and clean reads were mapped to the A. thaliana TAIR 10 genome using Bisulfite Sequence Mapping Program (BSMAP) with parameters “-v 2 -S 1” which allowing 2 mismatches. The methratio.py script from BSMAP with parameters “-r -z -p -m 1” was used to extract the methylation ratio from mapping results; only mapped reads after deduplication were considered for subsequent analyses.
For ChIP-seq data analysis, around 15.2 million raw paired-end reads were obtained for each sample and subsequently cleaned by Trimmomatic. Clean reads were mapped to the reference genome by Bowtie2 using the parameter "--very-sensitive --no-unal --no-mixed --no-discordant -k 2". Subsequently, uniquely mapped reads were selected and marked as duplicates using the Picard tool followed by using the SAMtools “rmdup” command. Coverage of deduplicated reads was normalized to 1 × sequencing depth by bamCoverage in deepTools with parameters "--normalizeUsing RPGC --exactScaling -bs 10".
Identification of non-reference TE insertions with TSDs (Target Site Duplications) was conducted using SPLITREADER with some modifications.
Genome_build: TAIR10
Supplementary_files_format_and_content: methylation wig files, RNA-seq coverage bedgGraph, ChIP-seq BigWig files
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| Submission date |
Feb 01, 2021 |
| Last update date |
Aug 11, 2021 |
| Contact name |
Cheng Zhao |
| E-mail(s) |
zhaocheng3326@gmail.com
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| Organization name |
Karolinska institute
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| Department |
Clinical Science
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| Lab |
Petropoulos lab
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| Street address |
H9 Klinisk vetenskap,171 77 Stockholm
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| City |
Stockholm |
| State/province |
Stockholm |
| ZIP/Postal code |
171 77 |
| Country |
Sweden |
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| Platform ID |
GPL17639 |
| Series (1) |
| GSE165877 |
Pathway conversion enables a double-lock mechanism to maintain DNA methylation and genome stability |
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| Relations |
| BioSample |
SAMN17694851 |
| SRA |
SRX9977522 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5051974_ddm1nrpd1_rep2.bedgraph.tdf.gz |
51.2 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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