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Sample GSM2211045

Query DataSets for GSM2211045

Status

Public on Jul 15, 2016

Title

MYB93R3

Sample type

SRA

Source name

Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens transformed with P103(35::MYB93) and pBIN61(P19)

Organism

Nicotiana benthamiana

Characteristics

tissue: leaves
transformant: 35S::MYB93/pBIN(p19)

Treatment protocol

N/A

Growth protocol

The two Agrobacterium tumefaciens GV3101-pMP90 strains (p103::MDP0000320772 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/mL). The cultures in 100 mL Erlenmeyer flasks were agitated at 130 rpm/28°C for 48 h. After centrifugation (4000 rpm/10 min), cultures were re-suspended in 20 mM MES, 20 mM MgSO4 infiltration buffer supplemented with 150 mg/mL acetosyringone. After 2 hours, p103::MDP0000320772 and pBIN61-P19 A. tumefaciens cultures were adjusted to OD600=0.8 and 1, respectively. Three-weeks old N. benthamiana plants were used for the transient expression study. Plants were divided into two groups, p103::MDP0000320772/pBIN61-p19 and pBIN61-P19(control), each with 4 biological replicates constituted by a pool of 6 plants. For each plant, the four first leaves were infiltrated with 0.5mL infiltration buffer using a 1 mL needleless syringe. After 6 days, the leaves were collected and flash frozen in liquid nitrogen.

Extracted molecule

total RNA

Extraction protocol

Total RNA from N. benthamiana leaves was extracted 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) was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA purity was assessed using a Nanodrop ND1000 spectrophotometer (Thermo scientific, Villebon-sur-Yvette, France). Total RNA was quantified using a Qubit RNA assay kit (Life technologies, Carlsbad, CA, USA).
cDNA libraries were prepared from 10ng 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. 2015. The pooled libraries were sequenced on an Illumina MiSeq using 5 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

Description

biological replicate 3

Data processing

The MiSeq generated FASTQ files were imported in CLC genomics workbench v6.5 discarding reads with poor quality (<Q30). Read duplicates were removed from each sample independently using the duplicate read removal plugin, Illumina adapter residues were then trimmed using the TruSeq adaptor sequence and finally filtered using the following criteria: low sequence quality <0.01, maximum of 2 ambiguous nucleotides, read length between 35 and 80 nucleotides.
FASTQ files were imported 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 (Nakasuki 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 (stringent mapping), mapped reads having more than 5 hits were removed reads should have 80% identity and 50% coverage to the reference transcriptome. The remaining unmapped reads were then re-mapped against the transcriptome v5-alternate transcripts following the same criteria. 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). Differentially expressed genes between the tobacco leaves infiltrated with A. tumefaciens GV3101 (pMP90, P103::MDP0000320772) and control were statistically determined using a Baggerley’s ‘on proportions’ weighted test (Baggerly et al. 2003), a false discovery rate corrected p-value (Benjamini-Hotchberg correction) was set at 0.01, a fold-change cut-off value set at 10-fold increase or decrease in expression, and a minimum mean RPKM difference between both groups set at 10.
Genome_build: 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; Log2 transformed fold change for each contig.

Submission date

Jun 22, 2016

Last update date

May 15, 2019

Contact name

Sylvain Legay

E-mail(s)

sylvain.legay@list.lu

Organization name

Luxembourg Institute of Science and Technology

Department

ERIN department