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| Status |
Public on Jul 18, 2016 |
| Title |
0-5 mm_rep1 |
| Sample type |
SRA |
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| Source name |
brown rot decayed wood
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| Organism |
Postia placenta |
| Characteristics |
strain: MAD698-R
wafer locus: 0-5 mm
decay stage: early decay
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| Growth protocol |
Postia placenta dikaryotic strain MAD 698-R (ATCC 44394) was maintained on 1.5% malt extract agar for routine culturing and to inoculate modified ATSM soil-block microcosms. Vertically-oriented wood wafers were colonized directionally from a pre-developed P. placenta hyphal mat. At harvest, wafers were sectioned to spatially segregate the temporal progression of wood decay, an approach modified from Schilling et al. (2013). In brief, a 1:1:1 mixture of soil, peat and vermiculite hydrated to 40-45% moisture content was packed into pint glass jars to one-third full. Feeder strips on the media surface were inoculated with 1-cm diameter agar plugs from P. placenta cultures and allowed to form a confluent mycelial lawn over 2-3 weeks. Wood wafers were cut 60 × 25 × 2.5 mm, with the largest face being the cross section and with the tangential plane in contact with the mycelium when propped in jars. Cultures were incubated at room temperature for 3-4 weeks until the visible hyphal front had advanced ~50 mm up the wafers. Wafers with flat hyphal fronts (horizontal lines) were then harvested, scraped free of surface hyphae, and sectioned into 5-mm sections using a razor blade sterilized with each cut, with the first cut along the hyphal front. Distance from the hyphal front was used to delineate sections on wafers for analyses per section in triplicate. The sections 0-5 mm, 15-20 mm and 30-35 mm were used for RNA-seq analysis.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA for P. placenta was isolated from each wood section with TRIzol (Life Technologies, Carlsbad, CA) and then cleaned up with a Qiagen RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) to remove organic inhibitors prior to downstream RNA-seq.
For RNA sequencing, TruSeq RNA v2 libraries were prepared and sequenced on a HiSeq 2500 System (Illumina Inc., San Diego, CA) by using the standard protocols from Illumina.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Temporal eparation of brown rot was sptially acomplished by wafer design
FPKM_tracking_0-5 mm, 15-20 mm and 30-35 mm_Cuffidff.txt
Paired_comparison_of_three_sections_Cuffdiff.xlsx
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| Data processing |
The RNA-seq data analyses were performed in the Galaxy platform (https://galaxy.msi.umn.edu/) according to the routine pipeline of Trapnell et al. (2014). Raw reads were first cleaned up with Trimmomatic (v0.3) by setting the parameters as: java -jar trimmomatic-0.30.jar PE --phred33 input_forward.fq.gz input_reverse.fq.gz output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP: TruSeq2-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. The qualities of the trimmed reads were further verified by FastQC (Galaxy Version 0.63). Then, the cleaned reads were mapped against the genome of P. placenta MAD 698-R (v1.0) (Postia_placenta.fasta) with Tophat (v2.0.13): tophat -r 0 -i 20 -I 5000 --min-segment-intron 20 --max-segment-intron 5000 -G Postia_placenta_FilteredModels2.gff -o tophat_out_? $INPUT/Pospl1 trimmomatic/Ppl_?.R1.PE.fastq trimmomatic/Ppl_?.R2.PE.fastq.
By using the reference transcript models Postia_placenta_FilteredModels2.gtf from JGI (http://genome.jgi.doe.gov/Pospl1/Pospl1.download.html), expression levels (FPKM, fragments per kilobase unique exon sequence per megabase of library mapped) were estimate by Cufflinks (Galaxy Version 2.2.1.0) with the classic FPKM normalization method.
By using the reference transcript models Postia_placenta_FilteredModels2.gtf from JGI (http://genome.jgi.doe.gov/Pospl1/Pospl1.download.html), expression levels (FPKM, fragments per kilobase unique exon sequence per megabase of library mapped) and significances of differences were also estimated with Cuffdiff (Galaxy Tool Version 2.2.1.3) using geometric normalization. Difference significances were calculated comparing each pairwise combination of the three section samples. The Cuffdiff output (e.g., all gene expression density distribution, principal component analysis and sample dendrogram for all gene expression) were visualized by cummerbund (Galaxy Version 1.0.1). Comparisons of gene expression from each two sections were presented as scatter plots by using RStudio (Version 0.99.491.).
Genome_build: Postia placenta MAD 698-R v1.0 from JGI (http://genome.jgi.doe.gov/Pospl1/Pospl1.download.html) [Postia placenta V1.0]
Supplementary_files_format_and_content: [.txt] Tab-delimited text files include FPKM values for each sample
Supplementary_files_format_and_content: [.xlsx] file includes pairwise comparison and FPKM levels
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| Submission date |
Jul 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jonathan Schilling |
| E-mail(s) |
schillin@umn.edu
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| Phone |
612-624-1761
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| Organization name |
University of Minnesota
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| Street address |
2004 folwell ave
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| City |
saint paul |
| State/province |
minnesota |
| ZIP/Postal code |
55108 |
| Country |
USA |
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| Platform ID |
GPL22164 |
| Series (1) |
| GSE84529 |
Localizing gene regulation by RNA-seq reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta |
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| Relations |
| BioSample |
SAMN05414141 |
| SRA |
SRX1959093 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2241531_0-5_mm_rep1_FPKM_Cufflinks.txt.gz |
545.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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