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Sample GSM6553958

Query DataSets for GSM6553958

Status

Public on Sep 26, 2022

Title

MYB52R1

Sample type

SRA

Source name

Leaves

Organism

Nicotiana benthamiana

Characteristics

tissue: Leaves
cell line: 4-week old
treatment: Infiltration with 35S::MdMYB52/pBIN(p19)

Treatment protocol

Transient expression were performed using four-weeks old N. benthamiana plants. Each biological replicate was constituted by a pool of 3 plants where the four first leaves were infiltrated with 0.5 mL infiltration buffer using a 1 mL needleless syringe. For the RNA-Seq experiment, plants were divided into two groups, p103::MD05G1011100/pBIN61-p19 (overexpression) and p103-empty/pBIN61-P19 (control) with 4 biological replicates each and were collected 4 days after infiltration. All samples were flash frozen in liquid nitrogen directly after collection.

Growth protocol

Three A. tumefaciens GV3101-pMP90 strains (p103::MD05G1011100, p103-empty and pBIN61-p19) were grown in 50 mL of LB liquid medium supplemented with gentamycin (30 mg/L), rifampicin (10 mg/L), kanamycin (50 mg/L), and acetosyringone (30 mg/L). As A. tumefaciens is not sensitive to the CcdB protein, we used the p103-empty vector as control in the present work (Traore and Zhao, 2011). The cultures in 100 mL Erlenmeyer flasks were agitated at 130 rpm/28°C for 48 h. After centrifugation (1000 g/10 min), cultures were re-suspended in infiltration buffer (20 mM MES, 20 mM MgSO4, 150 mg/L acetosyringone). After 3 hours, p103::MD05G1011100, p103-empty and pBIN61-p19 A. tumefaciens cultures were mixed and adjusted to OD600=0.8, 0.8 and 1, respectively.

Extracted molecule

total RNA

Extraction protocol

total RNA were extracted from two groups (p103::MD05G1011100/pBIN61-p19 (overexpression) and P103 (empty vector)/pBIN61-p19 (control)) of agroinfiltrated leaves. Three and 4 biological replicates were used for RT-qPCR and RNA-Seq, respectively. Each biological replicate was comprised of a pool of 3 plants with 4 agroinfiltrated leaves per plant. Total RNA extracts were obtained using the RNeasy plant mini kit (QIAGEN, Leusden, The Netherlands) coupled with on-column DNase I treatment, following the manufacturer’s guidelines. Total RNA integrity (RIN>8) and purity were assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a Nanodrop ND1000 spectrophotometer (Thermo scientific, Villebon-sur-Yvette, France), respectively. Total RNA was quantified using a Qubit RNA assay kit (Life technologies, Carlsbad, CA, USA). For gene isolation and RT-qPCR, reverse transcription was carried out as described in our previous work (Legay et al., 2015).
cDNA libraries were prepared from 20 ng mRNA using the SMARTer Stranded RNA-Seq kit according to the manufacturer’s guidelines (ClonTech laboratories, Mountain View, CA, USA). Libraries were analyzed and quantified as described in Legay et al. (2016). The pooled libraries were sequenced on an Illumina MiSeq using 4 consecutive runs (Illumina MiSeq reagent V3-150 cycles) to generate 76 base-pairs paired-end reads

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

FASTQ files were imported in pairs in CLC genomics workbench v8.0.1 discarding poor-quality reads (<Q30). For each library, reads were trimmed and filtered using the following criteria: sequence quality <0.01, no ambiguous nucleotides, minimum read length >35 nucleotides, trimming against the Illumina adaptor sequence, and finally a hard trim of 10 nucleotides at the 5’ end and 2 nucleotides at the 3’ end. The filtered reads were mapped to the N. benthamiana transcriptome v5-primary transcript (Nakasugi et al., 2014) obtained from the N. benthamiana genome and transcriptome website (http://benthgenome.qut.edu.au/) with the following criteria: a mismatch, gap and insertion cost on the maximum settings (3=stringent mapping), reads should have 80% identity and 90% coverage to the reference transcriptome. An additional annotation of the transcriptome was performed against the A. thaliana database using BLAST2GO Pro v3.0. Expression values were calculated using the RPKM (Reads per kilobase transcript per million reads) method (Mortazavi et al., 2008). In order to determine the differentially expressed genes between the tobacco leaves infiltrated with p103::MD05G1011100/pBIN61-p19 and p103-empty/pBIN61-P19, a Baggerley’s ‘on proportions’ weighted test (Baggerly et al., 2003) combined with a false discovery rate correction (Benjamini-Hotchberg) set at 0.05 was used. Expression cut-off values were set at 2-fold increase/decrease and 10 RPKM difference, respectively.
Assembly: N. benthamiana transcriptome v5-primary and alternate transcript (Nakasuki et al. 2014)
Supplementary files format and content: tab-delimited text files include, unique and toal hit reads, RPKM values for each Sample fold change for each contig.

Submission date

Sep 07, 2022

Last update date

Sep 26, 2022

Contact name

Sylvain Legay

E-mail(s)

sylvain.legay@list.lu

Organization name

Luxembourg Institute of Science and Technology

Department

ERIN department