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Sample GSM7851108

Query DataSets for GSM7851108

Status

Public on Feb 23, 2024

Title

12DPA_16C_biorep3

Sample type

SRA

Source name

tomato pericarp

Organism

Solanum lycopersicum

Characteristics

tissue: tomato pericarp
cell type: 16C sorted nuclei
genotype: WVA106
sample type: nuclei sorted by ploidy level
developmental stage: 12DPA
ploidy: 16C

Growth protocol

Tomato plants Solanum lycopersicum Mill. Cv. West Virginia 106 (WVA106) were cultivated in greenhouse with photoperiod of 16h light condition at 25°C and 8h night at 20°C and a relative humidity between 70 and 75%. Individual flowers were tagged on the day of anthesis, fully opened flowers that is wilt the next day. Fruits were harvested at three stages: 6DPA,9DPA and 12DPA.

Extracted molecule

nuclear RNA

Extraction protocol

Nuclei were prepared from 20 tomato fruits pericarp at 6DPA and 10 fruits at 9DPA and 12DPA. Nuclei were sorted by FANS as described in (Bourge et al., 2018). Pericarp pieces were chopped using a razor blade in 2mL of Gif Nuclear Buffer (GNB: 45 mM MgCl2, 30 mM Sodium-Citrate and 60 mM MOPS pH 7.0, 1% PVP 10.000, 0.1% Triton X-100 and 10 mM sodium metabisulfite (S2O5Na2)) and the suspension was filtered twice through a 48 µm nylon mesh before adding DAPI (4’,6-diamidino-2-phenylindole). 100k nuclei were sorted according to their ploidy levels with a MoFlo ASTRIOS EQ (BECKMAN COULTER) and collected in a tube containing 900µL of TRIzol™ Reagent (THERMOFISHER SCIENTIFIC). Before RNA extraction, 1µL of a dilution 1:1000 of ERCC RNA Spike-In Mix (THERMOFISHER SCIENTIFIC) was added. The mix of nuclei and spike-ins were shortly vortexed and rested for 5mn at room temperature before adding 200µL of Chloroform, agitating the tubes by hand for 15sec and incubating them for 3min at room temperature. After the incubation, tubes were centrifuged for 15mn at 12000g and 4°C, and the upper aqueous phase was transferred (around 840µL) to a fresh tube and mixed with 420µL of 100% ethanol. Then, total RNA was further purified using the RNeasy Micro Kit (QIAGEN) according to the provider’s instructions. A final volume of 14µL of total RNA was obtained.
Total RNA was used for construction of libraries following the instructions from the Illumina TruSeq Stranded RNA Sample Prep Kit. After library preparation for sequencing, a Duplex-specific nuclease (DSN) treatment was done using the commercial enzyme from EVROGEN following manufacturer’s instructions. Following DSN treatment, libraries were purified with SPRI beads (BECKMAN) and paired-end sequenced (2x150bp) with a HiSeq3000 (ILLUMINA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

D12.R3.16C

Data processing

Sequence quality was controlled with FastQC, read trimmed with cutadapt (https://doi.org/10.14806/ej.17.1.200) and mapped with STAR (Dobin et al., 2013), as described in Pirrello et al., 2018.
We used the SLYMIC 1.0 reference genome along with ERCC sequences for read mapping with STAR (Dobin et al., 2013). After mapping, paired reads were assigned to genes or ERCC spike-ins using featurecounts from the subread package (Liao et al., 2014) and SLYMIC 1.0 annotation.
Differential gene expression analyses were performed with the DESeq2 package (Love et al., 2014), after standard normalization, in R/Bioconductor as previously described (Pirrello et al., 2018). We considered as differentially expressed the genes that displayed an absolute log2 fold-change > 1 and an FDR-adjusted p-value <0.01.
Assembly: SLYMIC 1.0
Supplementary files format and content: rawCounts.tsv is a tab-separated matrix of read counts per gene obtained from featurecounts (for each sample: number of reads mapped to each gene)

Submission date

Oct 20, 2023

Last update date

Feb 23, 2024

Contact name

Pascal GP Martin

E-mail(s)

pascal.martin@inrae.fr

Organization name

INRAE

Department

UMR1332 BFP