NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1701496 Query DataSets for GSM1701496
Status Public on Sep 16, 2015
Title 96h rep1 barcode1
Sample type SRA
 
Source name fungal colonized wood section_96hrs
Organism Phanerodontia chrysosporium
Characteristics strain: RP78
time point: 96 hrs after inoculation
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
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description run217.2-96_ACTTGA_L003_R1
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.
 
Submission date Jun 02, 2015
Last update date May 15, 2019
Contact name Dan Cullen
E-mail(s) dcullen@wisc.edu
Phone 608-231-9468
Organization name UW/FPL
Street address One Gifford Pinchot Dr
City Madison
State/province WI
ZIP/Postal code 53726
Country USA
 
Platform ID GPL20273
Series (1)
GSE69461 Regulation of gene expression during the onset of ligninolytic oxidation by Phanerochaete chrysosporium on colonized wood
Relations
BioSample SAMN03755350
SRA SRX1045766

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

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap