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Sample GSM1657820 Query DataSets for GSM1657820
Status Public on May 16, 2016
Title Mix Day 4 Rep 3
Sample type SRA
Source name Mixed species soil sample
Organisms Sclerotinia sclerotiorum; Trichoderma hamatum GD12
Characteristics isolate: GD12 & M448
Treatment protocol Soils were supplemented with one of the following treatments; T. hamatum GD12, S. sclerotiorum, both fungal treatments or no treatment (control). T. hamatum GD12 was prepared as a bran inoculum by culturing five plugs taken from the leading edge of a three day old T. hamatum GD12 plate in a mixture of sterile 10 g bran + 30 ml water for 5 days at 26˚C under a 16 h light regime. The soil was inoculated with this mixture at a ratio of 1.3 g for every 50 g of soil (2.6% w/w). S. sclerotiorum was prepared as a poppy seed inoculum by culturing 10 g poppy seeds (sterilised as a mixture with 10 ml water) with 10 1 mm plugs for 10 days at 26˚C under a 16 h light regime before incorporating into soil at 2.6% (w/w) Soil from 2 wells of the same treatment were combined, snap frozen in liquid nitrogen and stored at -80˚C at six timepoints over a time course of 15 days (1d, 2d, 4d, 7d, 10d, 15d).
Growth protocol 50 g of sieved sphagnum moss peat (Shamrock) was supplemented with a treatment and split between the wells of a 6 well plate. Two lettuce seeds were sown into each well. The first seedling to emerge was preserved and the second removed upon emergence. Plants were watered with sterile water.
Extracted molecule total RNA
Extraction protocol 2 g of soil from each sample was used for RNA extraction. Samples were prepared for RNA extraction by homogenisation in a blender in the presence of liquid nitrogen. RNA was extracted using RNA PowerSoil® Total RNA Isolation Kit (Cambio UC-12866-25). Remaining soil was stored at -80˚C.
2 µl of diluted ERCC RNA control spike-in mix 1 or control spike-in mix 2 (Life Technologies) was added to 1 µg of sample RNA and the volume adjusted to 50 µl. Sequencing Libraries were prepared using the Maestro-based TruSeq RNA application from Perkin Elmer for Illumina TruSeq RNA library preparation on a Sciclone NGS platform. Samples were processed in 96 well hard shell PCR plates (Biorad HSP-9631). Washes were done in 150 µl bead washing solution instead of the 200 µl used in the manual process. Incubations at 65°C and 16°C for mRNA denaturation, and second-strand synthesis were performed on the Sciclone deck. However, the 80°C and 94°C incubations for mRNA elution and fragmentation, and first-strand cDNA synthesis reactions were performed on a thermocycler with heated lid to avoid excess evaporation. Purification and size selection was by Ampure XP bead clean up rather than gel based size selection. Library quality and quantity was determined by LabChipGX high sensitivity assay, after diluting the libraries 2µl in 20 µl.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Description processed data column: 4_9
Data processing Base Calling carried out using CASAVA v1.8.2
Fastq-Mcf was used to remove adapter sequences and trim reads of a phred quality score < 20 with the following parameters: -q 20 -l 35 -k 0 -p 15 --max-ns 0 -m 3. ERCC reads were removed using Bowtie v1.0.0 with the following parameters: -S -X 600
Reads were aligned to the reference genomes for Trichoderma hamatum GD12 and Sclerotinia sclerotiorum isolate 1980 using TopHat v2.0.8b with the following parameters: -r 200 --mate-std-dev 375 --library-type fr-unstranded.
Reads were quantified at each predicted gene location using htseq-count v0.6.0 with the following parameters: -m union -s yes
DESeq2 was used to calculate normalised counts and identify genes with statistically significant differential expression.
Genome_build: Trichoderma hamatum GD12 and Sclerotinia sclerotiorum 1980
Supplementary_files_format_and_content: Tab delimited matrix containing the normalised count for each gene in each sample.
Submission date Apr 15, 2015
Last update date May 15, 2019
Contact name Sophie Shaw
Organization name University of Aberdeen
Department Centre for Genome Enabled Biology and Medicine
Street address 23 St. Machar Drive
City Aberdeen
State/province Aberdeen City
ZIP/Postal code AB24 3RY
Country United Kingdom
Platform ID GPL20048
Series (1)
GSE67909 Transcriptional reprogramming underpins enhanced plant growth promotion by the biocontrol fungus Trichoderma hamatum GD12 during antagonistic interactions with Sclerotinia sclerotiorum in soil
BioSample SAMN03486615
SRA SRX996806

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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