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Sample GSM6809668

Query DataSets for GSM6809668

Status

Public on Jan 23, 2023

Title

Empty vector-4

Sample type

SRA

Source name

Leaves

Organism

Nicotiana benthamiana

Characteristics

tissue: Leaves
age: 4-week old
treatment: Infiltration with EmptyVector/pBIN(p19)

Growth protocol

A. tumefaciens GV3101-pMP90 strains (p103::MD08G1076200, 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). Cultures in 100 mL Erlenmeyer flasks were agitated at 130 rpm/30°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::MD08G1076200, p103-empty and pBIN61-p19 A. tumefaciens cultures were mixed and adjusted to OD600=0.8, 0.8 and 1, respectively. For the RNA-Seq, qPCR validation, cell wall analysis and lipid analysis, independent transient expression experiments have been performed using p103:: MD08G1076200/pBIN61-p19 and p103-empty vector/pBIN61-P19 A. tumefaciens mixtures using four biological replicates (n=4). Each biological replicate is constituted by three plants and four leaves per plants.

Extracted molecule

total RNA

Extraction protocol

total RNA were extracted from two groups (p103:: MD08G1076200 /pBIN61-p19 (overexpression) and P103 (empty vector)/pBIN61-p19 (control)) of agroinfiltrated leaves. Four biological replicates were used for RNA-Seq. 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).
cDNA libraries were prepared from 3 µg total RNA of each replicate according to the KAPA HyperPrep Kit manufacturer’s guidelines (Roche, Basel, Switzerland). All libraries were analyzed and quantified as described in Legay et al. (2016). The pooled libraries were sequenced on an Illumina NextSeq500 (NextSeq 500/550 Mid Output Kit v2.5 (150 Cycles)) to generate 75 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 v11.0.1 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 14 nucleotides at the 5’ end and 3 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 set at 2,3 and 3 respectively (3=stringent mapping), reads should have 80% identity and 80% coverage with the reference transcriptome.
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

Dec 12, 2022

Last update date

Jan 23, 2023

Contact name

Sylvain Legay

E-mail(s)

sylvain.legay@list.lu

Organization name

Luxembourg Institute of Science and Technology

Department

ERIN department