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Sample GSM4188541

Query DataSets for GSM4188541

Status

Public on Mar 06, 2020

Title

35S:A9 transgenic seedlings

Sample type

SRA

Source name

Seedlings

Organism

Nicotiana tabacum

Characteristics

tissue: Seedlings
time: 3-weeks old
genotype/variation: 35S:A9

Treatment protocol

For sequencing, which was performed twice for each RNA sample, we used in each case equimolecular mixtures of 4, replicate RNA samples were prepared from corresponding pairs of transgenic (T: 35S:A9, DS10:A9 or DS10:A4a), and sibling non-transgenic (NT) lines

Growth protocol

Tobacco (Nicotiana tabacum L. var. Xanthi) was used for all experiments. Seed sterilization, germination and seedling growth under controlled photoperiodic illumination conditions of 16 h light/8h dark, and 100 µmol m-2 s-1 fluence intensity for white-light, were as previously described (Prieto-Dapena et al., 2017 and references therein).

Extracted molecule

total RNA

Extraction protocol

Total RNA samples were prepared by the LiCl method from 3-week old seedlings (in the 35:A9 experiment), or from seeds (in the DSA10:A9 and DS10:A4a experiments)
Library preparation and sequencing on an Illumina HiSeq 4000 sequencer using 100-bp paired-end reads protocol were performed by BGI Co., Ltd (https://en.genomics.cn/). Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR were used in quantification and in qualification of the sample libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Sequenced reads were trimmed to remove adaptor sequences, and masked to remove low-complexity or low-quality sequence (reads in which N bases were more than 5% abundant, or base quality was lower than 15 for more than 20% of the bases were removed)
The sequenced "clean reads" from each sample (at least 4.44 Gb bases) were mapped to a Nicotiana tabacum reference genome using HISAT (v0.1.6-beta) with default parameters except for: --phred64 --sensitive --no-discordant --no-mixed -I 1 -X 1000
Novel transcripts prediction was performed using StringTie (v1.04) with default parameters except for: -f 0.3 -j 3 -c 5 -g 100 -s 10000 -p 8
CuffCompare (v2.2.1) with default parameters except for -p 12 was used to compare reconstructed transcripts to reference annotation, and coding potential of novel transcripts ('u','i','o','j' CuffCompare class code types) was predicted using CPC (v0.9-r2) with default parameters
The coding novel transcripts were merged with the reference Nicotiana tabacum transcriptome to get a complete reference to be used in gene expression analyses
Clean reads were mapped to the complete reference using Bowtie2 (v2.2.5) with default parameters except for -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200
Gene expression levels for each sample were calculated with RSEM (v1.2.12) with default parameters
Using PoissonDis (Poisson Distribution), the expression results were analyzed to detect Differentially Expressed Genes (DEG) between the corresponding pairs of non-transgenic and transgenic samples. We considered genes with a Fold Change (Log2) > 1.5 as DEG
Genome_build: Nicotiana tabacum (Ntab-BX_AWOK-SS.fa.gz/Ntab-BX_AWOK-SS_Basma.mrna.annot.fna)
Supplementary_files_format_and_content: Processed tables showing the functional annotation of genes, the gene expression levels per condition and the output of the differential gene expression analyses bvetween each pair of conditions

Submission date

Nov 22, 2019

Last update date

Mar 06, 2020

Contact name

Jose Luis Ruiz Rodriguez

Organization name

IPBLN-CSIC

Street address

Avda. del Conocimiento 17. P. T. Ciencias de la Salud

City

Granada