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Sample GSM4614412

Query DataSets for GSM4614412

Status

Public on Oct 23, 2020

Title

wt_heat_rep1

Sample type

SRA

Source name

Seedling

Organism

Arabidopsis thaliana

Characteristics

ecotype: Columbia
genotype: WT
treatment: Heat treated
biological replicate: 1

Treatment protocol

For heat stress treatments we incubated 10-day-old seedlings at 4 °C for 1h followed by 37 °C for 24h in the dark, as previously published (Shen et al., 2014).

Growth protocol

Plants were grown on plates with ½ MS medium including vitamins (Duchefa, Haarlem, The Netherlands) and 1% sucrose. Zebularine treated plants were grown on ½ MS medium supplemented with 40 um zebularine (Abcam). Plates were sealed with micro-pore tape and stratified for two days at 4°C in the dark. The plates were then transferred to a growth chamber with 16 h light (110 µmol m-2 s-1, 22°C) and 8 h dark (20°C) periods. Ten-day-old seedlings were used for RNA extraction.

Extracted molecule

genomic DNA

Extraction protocol

RNA extraction of three biological replicates (50mg plant material per replicate) was performed using an RNeasy Plant Mini Kit (Qiagen).
Genomic DNA was extracted using a MagJET Plant Genomic DNA Kit (K2761).
RNA seq libraries were generated using DNA-free RNA with the TruSeq RNA Library Prep Kit v2 (Illumina) according to manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq2000 in 50-bp single-end mode.
BSeq libraries for two replicates were prepared by Novogene (Hongkong, China) and sequenced on an Illumina HiSeq2000 in 150-bp paired-end mode.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina HiSeq 2500

Description

10 days old seedlings
wt_heat_MX_CG.txt.gz
wt_heat_MX_CHG.txt.gz
wt_heat_MX_CHH.txt.gz

Data processing

Quality control was done with an in-house R script
Bisulfite-seq: The 150 bp long pair-end reads were first quality trimmed by removing the first 5 bases from the 5' end and the last 20 bases from the 3' end. Reads were mapped to the reference genome TAIR10 in PE mode (--score_min L,0,-0.6) using Bismark v0.16.3. Mapped reads were deduplicated and cytosine methylation values calculated using the Bismark Methylation Extractor. CX methylation reports were pooled for both replicates for further analyses.
RNA-seq: Untrimmed reads were mapped to the TAIR10 Arabidopsis reference genome using STAR (v2.5.3.a, (Dobin et al., 2012) ). Expression counts were generated using the R function summarizeOverlaps from the package HTSeq in union mode on exons from the reference transcriptome AtRTD2 (Zhang et al., 2017) . Differential expression analyses were performed using the R package DESeq2 [v1.20.0, (Love et al., 2014) ]. Genes with absolute log2FoldChange ≥ 1 and FDR ≤ 0.05 were considered as differentially expressed, using either genotype or unstressed condition and the control. Similarly, expression counts along TEs were generated using the R function summarizeOverlaps from the package HTSeq in union mode along the coordinates of the 31,189 TAIR10 annotated TEs. Differential expression analyses were performed using DESeq2 and the same threshold values as for gene transcripts. Subsequent analysis were performed in R. The gene and transposable element models used were from TAIR10 gff annotation file
Genome_build: TAIR10
Supplementary_files_format_and_content: Bisulfite-seq: Three genome-wide cytosine methylation reports (for CG, CHG, CHH) per condition were created. These reports have the following format: <chromosome> <position> <strand> <count methylated> <count unmethylated> <C-context> <trinucleotide context>
Supplementary_files_format_and_content: For the RNA-seq, tables with the values of the differential expression analysis for genes and TEs are presented in text files

Submission date

Jun 12, 2020

Last update date

Oct 23, 2020

Contact name

Minerva Susana Trejo Arellano

E-mail(s)

Minerva.Trejo-Arellano@jic.ac.uk

Organization name

SLU

Department

Plant Biology