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Sample GSM6606987

Query DataSets for GSM6606987

Status

Public on Oct 03, 2022

Title

C1_2

Sample type

SRA

Source name

cotyledons (1mm radicle seed)

Organism

Medicago truncatula

Characteristics

tissue: cotyledons (1mm radicle seed)
developmental stage: 1mm germinating seed
cultivar: A17
treatment: PEG-untreated

Treatment protocol

For the PEG treatment, 1mm and 5mm radicle seeds were subjected to a mild osmotic stress (-1.7 MPa) using a polyethylene glycol (PEG8000) solution for 3 days at 10°C dark (called 1mmPD and 5mmPD samples), then were washed and underwent desiccation as previously described .

Growth protocol

The seed germination was accessed by germinating triplicates of 50 dried seeds on Whatman paper No1 imbibed with 1ml of autoclaved water in 3 cm diameter Petri dishes at 20°C under a 16h/8h photoperiod for eight days. Then, seeds exhibiting a 1 mm (called 1mmD sample) and 5 mm (5mmD sample) radicles were sampled and used to perform a DT assay (Buitink et al., 2003), which consisted in slowly desiccating germinating seeds using a saturated solution of K2CO3 at 20°C for 72 hours.

Extracted molecule

total RNA

Extraction protocol

For Medicago early post-germination seeds, samples of radicles and cotyledons were used separately for RNA extractions. Samples were named accordingly: desiccated radicles of 1mm germinating seeds were called R1 and R1P (when subjected to PEG treatment), desiccated radicles of 5 mm germinating seeds were called R5 and R5P (when subjected to PEG treatment). About 100 freshly harvested seeds in two biological replicates were used to extract RNA for each sample. All samples were ground using micropestles and liquid nitrogen and RNA was extracted using the NucleoSpin® RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) with lysis buffer containing 1% of polyvinylpyrrolidone (PVP-40) followed by incubation at room temperature for 10 minutes.
All samples with good qualities (260/280 and 260/230 absorbance ratio >1.8; RNA Integrity Number, RIN>7; 28S/18S>1.7) were sent to Beijing Genomics Institute (https://www.bgi.com) (Hong Kong) for library preparation and sequencing on BGISEQ-500 platform, generating an average of 24M reads of 50bp per sample (20M SE50).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

C1_2

Data processing

The sequencing data was filtered with SOAPnuke (v1.5.2) [1] by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
SOAPnuke: version：v1.5.2, parameters: -l 15 -q 0.5 -n 0.1, website: https://github.com/BGI-flexlab/SOAPnuke
Quality control was checked using FastQC
high quality reads were mapped on Medicago truncatula (Mtv5) reference transcriptome using quasi-mapping alignment and quantification methods of Salmon algorithm v.1.2 (Patro et al., 2017)
Assembly: Medicago truncatula (Mtv5) from https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/ (file MtrunA17r5.0-ANR-EGN-r1.6.mrna.fasta)
Supplementary files format and content: tab-delimited text files corresponding to the Count table of all samples (columns) and all transcripts annotated in Mtv5 gff r1.6 (rows) (= count.txt)

Submission date

Sep 29, 2022

Last update date

Oct 03, 2022

Contact name

Jerome Verdier

E-mail(s)

Jerome.verdier@inrae.fr

Organization name

INRAE / IRHS

Lab

SEED