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Sample GSM5965223

Query DataSets for GSM5965223

Status

Public on Mar 25, 2022

Title

puchi_24h_R2

Sample type

SRA

Source name

puchi-1_24h post gravistimulation_R3

Organism

Arabidopsis thaliana

Characteristics

genotype: Mutants puchi-1
treatment: 24h gravistimulation
tissue: Lateral root and surrounding tissues at the bend
age: 4 day-old seedlings

Treatment protocol

Gravistimulation were performed on 4 day-old seedlings and root bends were sampled at 12 hours (h), 18h, 24h, 30h and 36h after gravistimulation. Three biological replicates (for both Col-0 and puchi-1) were used for the RNAseq experiment.

Growth protocol

Col-0 and puchi-1 seeds were surface-sterilized and sown on ½ strength Murashige and Skoog (½ MS) solid medium containing 0.7% (w/v) plant agar supplemented with B5 vitamins. Plates were kept at 4°C for 2 days and then placed in continuous light conditions (24-h light/0-h dark cycle) in vertical position.

Extracted molecule

total RNA

Extraction protocol

For each replicates, root bends of more than 400 seedlings were microdissected under a binocular microscope and frozen in liquid nitrogen immediately on harvesting. For reference, approximately 400 mature root segments located between the bend and the shoot were harvested in 4d-old seedlings at 9 h after an inductive gravitropic stimulation to be used as a reference of non-gravitropic- stimulated root tissues devoid of developing LRP (termed “time point 0” in the data set).Total RNA was extracted using Qiagen RNeasy plant mini kit with an on-column DNAse treatment following the manufacturer’s recommendation (RNAse-free DNAse Set, Qiagen, Crawley, UK). RNA samples were quantified using a Nanodrop ND100 spectrophotometer (Nanodrop, Wilimington, USA) and RNA purity and integrity were evaluated using High-Resolution Automated Electrophoresis 2100 Bioanalyzer system from Agilent Technologies (https://www.agilent.com/en/product/automated-electrophoresis).
cDNA Libraries were constructed using Stranded mRNA Prep Ligation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions and sequenced on a complete S1 flow cell (2 lanes) of an Illumina NovaSeq 6000 in paired-end 100 nucleotides mode.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Image analyses and base calling were performed using the NovaSeq Control Software and Real-Time Analysis component (Illumina). Demultiplexing and trimming were performed using Illumina's conversion software (bcl2fastq 2.20). The quality of the raw data was assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer).
A splice junction mapper, TopHat 2.1.1 (using Bowtie 2.3.5.1), was used to align the RNA-Seq reads to the Arabidopsis thaliana genome (NCBI TAIR10.1) with a set of gene model annotations (gff file downloaded from NCBI on November 13, 2020).
Final read alignments having more than 6 mismatches were discarded. Samtools (v1.9) was used to sort the alignment files. Then, the counting was performed with Featurecounts 2.0.0. As the data is from a strand-specific assay, the read has to be mapped to the opposite strand of the gene (-s 2 option).
Assembly: TAIR10.1
Supplementary files format and content: tab-delimited text files include raw gene counts for every gene (TAIR|NCBI GeneID|Symbol) and every sample

Submission date

Mar 22, 2022

Last update date

Mar 25, 2022

Contact name

Soazig GUYOMARC'H

E-mail(s)

soazig.guyomarch@ird.fr

Organization name

University of Montpellier

Lab

Plant Diversity, Adaptation and Development (DIADE), IRD