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| Status |
Public on Nov 27, 2018 |
| Title |
mRNA WT primed rep 3 |
| Sample type |
SRA |
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| Source name |
3 week old Col-0 leaf tissue
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: true leaves 4 - 9
ecotype: Col-0
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| Treatment protocol |
For excess-light treatments, exposure to approximately 10X growth irradiance (1000 μ mol photons m-2 s-1) was applied using a mixture of 250W metal halide lamps (Venture Lighting, MH 250W/U) and high pressure sodium lamps (Phillips, SON-T 250W E E40 SL/12) providing a source of ‘warm’ light (similar to sunlight). This was applied for one hour repeated thrice daily at 9:30 am, 1:30pm, and 5:30 pm.
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| Growth protocol |
Plants grown on soil, with approximately 3g/L osmocote fertilizer, under a 12-hour photoperiod of 100-150μ mol photons m-2 s -1, 20 (±0.5) °C, and 55 (±5) % RH.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Bisulfite-seq: Genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit (Limburg, Netherlands), as per the manufacturer’s instructions. 100-200 ng of fragmented (Covaris) and purified gDNA was bisulfite converted using the Zymo DNA-Gold bisulfite conversion kit (Zymo Research; CA, USA).
mRNA-seq: Total RNA was extracted using TRIzol reagent.
Whole genome bisulfite sequencing libraries were constructing using the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences; MI, USA) as per the manufacturer’s instructions.
PolyA-enriched RNA-sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina, CA, USA) using 1.3 μg input of extracted total RNA. The following modifications to the manufacturer’s instructions were made: reagent volumes were adjusted for 1/3 reactions; and Invitrogen SuperScript III Reverse Transcriptase (Invitrogen, Life Technologies Australia Pty Ltd) was used for first strand synthesis with adjusted reaction temperature of 50 °C.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Bisulfite-seq
Raw reads were quality controlled using FastQC (v0.11.2), Cutadapt (v1.9), Trim Galore! (v.0.3.7).
Single-end alignments of trimmed raw reads were aligned to the TAIR10 reference using Bismark (v0.14.5) and Bowtie2 (v2.2.9) with the flags -N 0 and -L 20.
Per cytosine methylation levels were calculated using Bismark methylation extractor with default settings. Bisulfite conversion efficiency was calculated as the proportion of methylated cytosines in the CHH context within the chloroplast genome, which itself should be fully unmethylated.
Weighted methylation levels were used to calculate the proportion of CG, CHG, and CHH at single cytosine resolution to account for sequencing depth. This output was utilized in DMR identification using DSS.
mRNA-seq
Raw reads were diagnosed using FastQC (v0.11.2). Due to strong nucleotide sequence content bias Trim Galore! and Cutadapt were used to trim low-quality reads with PHRED score < 20 (-q 20) and to make a hard clip of 10 bp and 1 bp from the 5’ and 3’ ends, respectively.
Single-end alignments of trimmed raw reads were aligned to the TAIR10 reference genome using Subread (v1.6.2) with the flags -t 0 and -u to report uniquely mapping reads, prior to sorting, indexing and compressing using Samtools (v1.2).
Transcript quantification was performed at the gene-level with the Araport11 annotation using featureCounts (with flag -s 2 for reverse strand specificity). This output was utilized in differential gene expression testing with edgeR.
Genome_build: TAIR10
Supplementary_files_format_and_content: .bed.gz: weighted methylation levels at single cytosines. Separate files have been produced for each sequence of DNA methylation: CG, CHG, and CHH; . counts: counts per gene-level feature across the Araport11 annotation for each sample.
Supplementary_files_format_and_content: bed: chromosome, start, end, proportion methylated, counts methylated, counts un-methylated, total counts
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| Submission date |
Oct 11, 2018 |
| Last update date |
Nov 29, 2018 |
| Contact name |
Diep R Ganguly |
| E-mail(s) |
dganguly@sas.upenn.edu
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| Phone |
+1 215-898-0808
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| Organization name |
University of Pennsylvania
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| Department |
Department of Biology
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| Lab |
Brian Gregory
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| Street address |
433 S University Ave
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19103 |
| Country |
USA |
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| Platform ID |
GPL19580 |
| Series (1) |
| GSE121150 |
Excess light priming in Arabidopsis thaliana genotypes with altered DNA methylomes |
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| Relations |
| BioSample |
SAMN10232917 |
| SRA |
SRX4866876 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3427002_P-r3.sorted_genes_ara11.counts.txt.gz |
496.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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