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Status |
Public on Sep 16, 2015 |
Title |
96h rep1 barcode1 |
Sample type |
SRA |
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Source name |
fungal colonized wood section_96hrs
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Organism |
Phanerodontia chrysosporium |
Characteristics |
strain: RP78 time point: 96 hrs after inoculation
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Growth protocol |
Phanerochaete. chrysosporium (strain RP-78) was grown as described previously on microtomed tangential sections of spruce sapwood (Picea glauca, 40 mm long, 10 mm wide, 40 µm thick, dry wt approx. 7 mg) that were embedded on glass cover slips in 90 µl of agar containing a nitrogen/mineral salts medium without nutrient carbon
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted from fungal colonized wood sections prepared as described above. Samples, comprised of 20 sections each, were harvested after 40h (3 samples) and 96 hr (3 samples), immersed in liquid nitrogen and stored at -80C until extraction. For total RNA purification, wood sections were ground in prechilled mortar and pestle in liquid nitrogen. Autoclaved and prechilled glass beads (0.5mm) were used during the grinding to make the wood sections into fine powder. Following repeated phenol;chloroform extractions, RNA was ethanol precipitated at -20°C overnight and further purified using the RNeasy mini kit (Qiagen, Valencia, CA). Total RNA was DNAase treated according to the manufacture’s protocol. Messenger RNA was purified from 1μg total RNA using poly-T oligo-attached magnetic beads. Double-stranded cDNAs were synthesized using SuperScript II (Invitrogen, Carlsbad, California, USA) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA polymerase I and RNAse H for removal of mRNA. Double-stranded cDNA was purified using Agencourt AMPure XP beads (Qiagen, Valencia, California, USA) as recommended in the TruSeq RNA Sample Prep Guide. cDNAs were end-repaired by T4 DNA polymerase and Klenow DNA polymerase and phosphorylated with T4 polynucleotide kinase. The blunt ended cDNA was purified using Agencourt AMPure XP beads. The cDNA products were incubated with Klenow DNA polymerase to add an ‘A’ base (Adenine) to the 3’ end of the blunt phosphorylated DNA fragments and then purified using Agencourt AMPure XP beads. DNA fragments were ligated to Illumina adapters having a single ‘T’ base (Thymine) overhang at their 3’end. The adapter-ligated products were purified using Agencourt AMPure XP beads. Adapter ligated DNA was amplified in a Linker Mediated PCR reaction (LM-PCR) for 15 cycles using PhusionTM DNA polymerase and Illumina's PE genomic DNA primer set and then purified using Agencourt AMPure XP beads. Quality and quantity of the finished libraries were assessed using an Agilent DNA1000 series chip assay (Agilent Technologies, Santa Clara, CA) and Invitrogen Qubit HS Kit (Invitrogen, Carlsbad, California, USA), respectively, and libraries standardized to 2 μM.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
run217.2-96_ACTTGA_L003_R1
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Data processing |
Cluster generation was performed using a TruSeq Single Read Cluster Kit (v3) and the Illumina cBot, with libraries multiplexed in three HiSeq2000 lanes (barcodes 1,2,3). Images were analyzed using CASAVA version 1.8.2 and FASTQ files generated. RPKM generated. DNAStar Inc (Madison, WI) modules SeqNGen and Qseq were used for mapping reads and statistical analysis. Final fastq files processed using DNASTAR modules SeqNGen and Qseq Genome_build: DoE Joint Genome Institute version 2.2 at http://genome.jgi-psf.org/pages/dynamicOrganismDownload.jsf?organism=Phchr2 Supplementary_files_format_and_content: RPKM values for each Sample. Values for 13602 transcript models (Seq_ID column) predicted by the DoE JGI automated pipeline. Ratios and probabilites computed for 40 hour versus 96 hour expression levels. Corresponding protein models and InterPro domains also listed.
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Submission date |
Jun 02, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Dan Cullen |
E-mail(s) |
dcullen@wisc.edu
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Phone |
608-231-9468
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Organization name |
UW/FPL
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Street address |
One Gifford Pinchot Dr
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City |
Madison |
State/province |
WI |
ZIP/Postal code |
53726 |
Country |
USA |
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Platform ID |
GPL20273 |
Series (1) |
GSE69461 |
Regulation of gene expression during the onset of ligninolytic oxidation by Phanerochaete chrysosporium on colonized wood |
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Relations |
BioSample |
SAMN03755350 |
SRA |
SRX1045766 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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