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| Status |
Public on Aug 01, 2014 |
| Title |
Dark replicate 1 |
| Sample type |
SRA |
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| Source name |
mycelium
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| Organism |
Neurospora crassa |
| Characteristics |
strain number: FGSC2489
light induction time point: 0 min
genotype/variation: WT
tissue: mycelia
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| Treatment protocol |
Cultures were exposed to white-light using cool white fluorescent bulbs (1200 lux) and cells were harvested in a darkroom at time 0 (dark), 15, 60, 120 and 240 min after light exposure.
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| Growth protocol |
200-ml of Bird’s Medium was inoculated to a final concentration of 107 conidia/ml and grown in the dark for 24 hrs, at 25oC with orbital shaking (150 rpm)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen mycelia using 1 g 180 °C autoclaved Zirconia/Silica Beads (Biospec Products, Inc 11079105Z) and a Mini-BeadBeater-8 with ice-cold 580 µl extraction buffer (100 mM Tris-HCl, pH 7.5; 100 mM LiCl; 20 mM DTT), 420 µL phenol, 420 µL chloroform, and 84 µL 10% SDS. Immediately after removing from -80 °C, mycelia were homogenized by 1 min beating in the bead beater. After 4 min end-over-end rotation, the homogenate was centrifuged at 12000 g at 4 °C for 1 min to separate phases. The aqueous phase was extracted with phenol/chloroform and chloroform and precipitated in 0.3 M NaOAc/ethanol. The pellet was washed twice with 70% ethanol (prepared with DEPC-treated water), briefly air-dried, and then was dissolved in filter-sterilized DEPC-treated water. Poly(A) mRNA was purified from total RNA using oligo-dT cellulose (SACHS and YANOFSKY 1991) for replicate 1 or Ambion Poly(A)Purist MAG kit of replicate 2, and further treated with Ambion Turbo DNase kit and Epicentre mRNA-Only kit.
cDNA was suspended in 300 µl TE in a 1.5 ml TPX micro tube (Diagenode) and sheared using the low energy cycle for 15 min (15 sec on/15 sec off) with a Diagenode Bioruptor whose water bath was maintained at 4°C with a recirculating chiller. Sheared cDNA was transferred to a 1.7 ml polypropylene microcentrifuge tube and precipitated in 0.3 M NaOAc/ethanol.The pellet was washed once with 70% ethanol, air-dried, and dissolved in 15 µl sterile water and stored at -20°C. RNA-Seq library was prepared from 1 µg of sheared DNA using Illumina TruSeq Sample Prep kit. Each sample was ligated to an indexing adapter and selectively enriched through a 10-cycle amplification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
HTS760
mRNA profiling
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| Data processing |
Base-calling software was RTA 1.13.48, CASAVA v1.8.2
Reads were aligned to the Neurospora genome with TopHat v2.0.5 (-a 5 -m 1 -i 30 -I 2000 --library-type fr-unstranded).
FPKM and fold-changes were calculated using the cuffdiff command from version 2 of the Cufflinks suite (-b -u).
Genome_build: Neurospora crassa genome assembly 10, Gene annotation 10.6
Supplementary_files_format_and_content: txt file with gene name in column 1 and FPKM in column 2
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| Submission date |
Dec 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Kristina M Smith |
| E-mail(s) |
smitkris@science.oregonstate.edu
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| Organization name |
Oregon State University
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| Department |
Biochemisty and Biophysics
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| Lab |
Freitag
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| Street address |
ALS2011
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| City |
Corvallis |
| State/province |
OR |
| ZIP/Postal code |
97331 |
| Country |
USA |
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| Platform ID |
GPL16164 |
| Series (1) |
| GSE53534 |
Genome-wide characterization of light-regulated genes in Neurospora crassa |
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| Relations |
| BioSample |
SAMN02483530 |
| SRA |
SRX396823 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1295595_HTS760.txt.gz |
58.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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