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Sample GSM1654402

Query DataSets for GSM1654402

Status

Public on Aug 28, 2015

Title

Balsam-mock-2

Sample type

SRA

Source name

Fully expanded leaf

Organism

Populus balsamifera

Characteristics

inoculation: mock-water
sample time: 0 days after inoculation

Treatment protocol

One monosporal Sphaerulina isolate from each species was utilized. Isolates were prepared from spore suspensions stored in 25% glycerol at -80ºC. To obtain conidia for foliar application, storage cultures were seeded to potato dextrose agar (PDA; Difco 213400) plates and grown at 22ºC in the dark for 8 days. Conidia were harvested by flooding plates (20 per isolate) with sterile distilled water, then filtered through 100-µl nylon mesh sterile Falcon® cell strainers (Fisher Scientific, Waltham, MA, USA), centrifuged at 4000 x g for 10 minutes, washed with 40 ml of sterile distilled water and resuspended in sterile distilled water to 1x105 per mL prior to application. Using an airbrush, each tree was sprayed with 10 mL of atomized spore suspension. Three trees from each species were sprayed with water to serve as controls.

Growth protocol

Trees were propgated in the greenhouse in 3 L pots with supplemented light at 16 h daylengths for 5 months. The five month old trees were transferred to Conviron growth chambers set at 22°C and 16h light with a photosynthetic flux of 90-100 µmol m-2 s-1 and incubated for 4 weeks for a final age of 6 months.

Extracted molecule

total RNA

Extraction protocol

RNA was also isolated from 100 mg of liquid nitrogen chilled homogenized leaf tissue using the RNeasy Plant Mini Kit (Qiagen) with in column RNase-Free DNase Set (Qiagen) .
RNA was extracted from each tree sampled and assayed for quality with a 2100 Bioanalyzer and Nanodrop 1000 spectophotometer. All RNA samples had a ratio of the absorbance at 260 and 280nm (A260/280) above 2.0 and were confirmed to be free of genomic DNA. Libraries for each RNA sample were prepared by Brian Boyle at ‘L'Institut de biologie intégrative et des systèmes’ (IBIS) at Université Laval, Quebec City, Quebec, Canada. Each cDNA library was barcoded and six samples were pooled together. TruSeq RNA Sample Preparation Kit v2 was used to prepare DNA for Illumina sequencing

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

AF20

Data processing

Illumina Casava1.8 software used for basecalling.
Quality checking: base call quality , consequtive homopolymer, Kmer frequency, base call frequency, overrepresented sequence and basic statisitics was performed in RobiNA - transcriptomics data preprocessor version 1.2.1
Adapter trimining, sliding window trimmer, read length filter to remove reads shorter than 20 bp for all samples. RobiNA - transcriptomics data preprocessor version 1.2.1
Read mapping was performed using Bowtie v1 to reference genome allowing 2 mismatches in a 28 bp seed legnth in RobiNA - transcriptomics data preprocessor version 1.2.1.
Gene expression analysis step - edgeR with BH corrected p-value cutoff 0.05, auto dispersion and min Log-fold change of 1 within RobiNA - transcriptomics data preprocessor version 1.2.1
Unassembled reads were pooled for samples of the same species and de novo assembled using CLC Genomics Workbench version 7 with default settings. Contigs less than 500 bp in legnth were discarded.
Read mapping was performed using Bowtie v1 to reference genome allowing 2 mismatches in a 28 bp seed legnth in RobiNA - transcriptomics data preprocessor version 1.2.1.
Gene expression analysis step - edgeR with BH corrected p-value cutoff 0.05, auto dispersion and min Log-fold change of 1 for samples mapped to lists of contigs
Genome_build: P. trichocarpa V3.0 210
Supplementary_files_format_and_content: Read count files were generated using Bowtie v1 in RobiNA 1.2.1 mapping to either the P. trichocarpa V3 genome of a list of de novo assembled contigs; Digital gene expression analysis files were prouced by edgeR in RobiNA 1.2.1; de novo asssembled contigs (fasta) for each poplar species were produced using CLC Genomics Workbench version 7 de novo assembler with reads that did not map to the P. trichocarpa v3 genome.

Submission date

Apr 09, 2015

Last update date

May 15, 2019

Contact name

Adam Foster

Organization name

Natural Resources Canada

Department

CFS

Lab

Armand Seguin