 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 20, 2021 |
| Title |
RNAseq_Col-0_flg22_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Whole seedlings
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
age: 2-week-old seedlings
ecotype: Col-0
genotype: wild type
treatment: 100 nM flg22, 2 hours
|
| Treatment protocol |
200 2-week-old seedlings per genotype and biological replicate were pre-conditioned to liquid 1/2 MS 24 hours before replacing medium with 100 mM flg22 in 1/2 MS (liquid) or 1/2 MS (liquid) as mock treatment for 2 hours
|
| Growth protocol |
Arabidopsis seeds were sterilised with diluted bleach in 70% EtOH for 7 minutes. Seedlings were grown in ½ Murashige and Skoog medium 0.5% Phytagel, long day photoperiod (16 h light)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
flg22-treated and mock-treated seedlings were harvested onto lab tissue before freezing inmediatelly in liquid nitrogen. Frozen tissue was thoroughly ground to fine powder in liquid nitrogen using a pre-chilled pestle and mortar. RNA was extracted with the NucleoSpin® RNA kit (Macherey-Nagel) starting from ~100 mg of powder, following manufacturer’s specifications. For MNase-seq experiments, 2 g of frozen powder were used for nuclei extraction with 10 mL of nuclei extraction buffer 1 (0.4 M sucrose, 10 mM Tris/HCl, pH 8.00, 10 mM MgCl2, 5 mM ß-mercapto-EtOH, 0.1 mM PMSF and Protease Inhibitor Mix P, 39103 Serva), filtering debris out through a 200 μm filter and centrifuging supernatant at 1000 g for 10 min at 4 °C. Nuclei pellet was washed in 5ml of nuclei extraction buffer 2 (25 mM Sucrose, 10 mM Tris/HCl pH 8.00, 10 mM MgCl2, 1% Triton X-100, 5 mM ß-mercapto-EtOH, 0.1 mM PMSF and Protease Inhibitor Mix P, 39103 Serva), mixed by vortex, filtered using a using a 60 μm filter and centrifuged at 1000 g for 10 min at 4 °C. Nuclei pellet was rinsed with Mnase buffer (10 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2, 0.15 mM Spermine and 0.5 mM Spermidine) and re-suspended in 250 μL of MNase buffer. DNA concentration was quantified with a NanoDrop, and samples were diluted to 400 ng/μL. 1 μl of 25 U/μL micrococcal nuclease (MNase) was added to 125 μL per sample and incubated at 37 °C for 10 minutes. To stop the reaction, 125 μL of Stop Buffer 2x (50 mM EDTA, 50 mM EGTA and 1% SDS) Tris/HCl pH 6.50 and 4 μL of proteinase K (stock 10 mg/ml) were added and incubated at 45 °C for 1h. Samples were purified with a QIAquick PCR Purification Kit (Qiagen) and eluted with 15 μL water. Eluate was loaded onto a 1% agarose gel (without loading buffer dye) and the lowest band (mono-nucleosomal DNA) was excised and gel-purified with QIAquick® Gel Extraction Kit (Qiagen).
RNA library prep was carried out with a #E7420 S/L NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (New England Biolabs) following manufacturer’s instructions. Agencourt® AMPure® XP Beads (#A63881, Beckman Coulter, Inc.) magnetic beads were used for RNA purification. Genomic libraries for MNase-seq were prepared starting from 50 ng of DNA per sample. DNA library prep was carried out with a NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (E7645S/L, NEB) following manufacturer’s instructions. Agencourt® AMPure® XP Beads (#A63881, Beckman Coulter, Inc.) magnetic beads were used for DNA purification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
For RNA-seq, STAR was used to generate genome indices from GTF genome from emsembl Genome assembly TAIR10 (STAR --runThreadN 4 --runMode genomeGenerate --sjdbOverhang 75) and map reads from raw fastq files (STAR --runThreadN 4 --readFilesCommand gunzip -c --outSAMtype BAM SortedByCoordinate) to generate sorted BAM files.
For RNA-seq sorted BAM files, Htseq-count through LiBiNorm was used to produce tab delimited count files (count --order=pos --minaqual=10 --mode=intersection-nonempty --idattr=gene_id --type=exon --stranded=reverse)
For MNase-seq, raw reads from fastq files were trimmed with Trimmomatic (trimmomatic SE -threads 8 -phred33 LEADING:5 TRAILING:5 MINLEN:30)
For MNase-seq, trimmed reads were mapped with bowtie2 (-p 8 --very-sensitive -x) to TAIR10 genome version and sort with samtools and create a bigwig file for easy visualization
Genome_build: TAIR10
Supplementary_files_format_and_content: RNA-seq data as tab-delimited text files with count values for each sample. MNase-seq data as bigWig files generated using samtools from sorted bam files
|
| |
|
| Submission date |
Apr 30, 2020 |
| Last update date |
Apr 20, 2021 |
| Contact name |
Lorenzo Concia |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
769929568
|
| Organization name |
École Normale Supérieure
|
| Department |
Institut de Biologie
|
| Lab |
Plant and Algal Genomics Lab
|
| Street address |
46 Rue d'Ulm
|
| City |
Paris |
| ZIP/Postal code |
75020 |
| Country |
France |
| |
|
| Platform ID |
GPL19580 |
| Series (1) |
| GSE149654 |
Effect of flg22 peptide over transcriptome and nucleosome landscape of Arabidopsis on Col-0 and chromatin remodelling ATPase EDA16 mutants |
|
| Relations |
| BioSample |
SAMN14779713 |
| SRA |
SRX8215124 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4506674_RNA_Col_flg_1Aligned.sortedByCoord.out.bam_LiBiNormCounts_rev.txt.gz |
129.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
