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Sample GSM7851078 Query DataSets for GSM7851078
Status Public on Feb 23, 2024
Title 6DPA_2C_biorep3
Sample type SRA
 
Source name tomato pericarp
Organism Solanum lycopersicum
Characteristics tissue: tomato pericarp
cell type: 2C sorted nuclei
genotype: WVA106
sample type: nuclei sorted by ploidy level
developmental stage: 6DPA
ploidy: 2C
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 D06.R3.02C
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
Lab FDFE
Street address 71 avenue Edouard Bourlaux
City Villenave d'Ornon
ZIP/Postal code 33140
Country France
 
Platform ID GPL23294
Series (1)
GSE245923 Ploidy-specific transcriptomes shed light on the heterogeneous identity and metabolism of developing pericarp cells
Relations
BioSample SAMN37909525
SRA SRX22159234

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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