|
| Status |
Public on Jan 22, 2015 |
| Title |
15kGy_irradiated |
| Sample type |
SRA |
| |
|
| Source name |
15kGy_irradiated
|
| Organism |
Deinococcus radiodurans R1 = ATCC 13939 = DSM 20539 |
| Characteristics |
treatment: 15kGy_irradiated
|
| Treatment protocol |
D. radiodurans cells were cultured to exponential (OD600 = 1) phase, packed, frozen with dry ice and transported in sterilized plastic bags for irradiation. Samples were thawed at room temperature at the radiation facility before irradiation. These samples were kept cold on wet ice (0°C) while irradiated with a 10 MeV, 18 kW LINAC b-ray source at the National Center for Electron Beam Research, Texas A&M University. Cell samples were subjected to sham and 15 kGy (250Gy/s) exposures. This initial high radiation exposure was designed to elicit a strong enough response that would allow detectable differential expression of sRNAs by Northern blotting. Cells were diluted 4-5 fold to OD600 =1 and recovered in fresh culture (TGY) medium for 2 hours at 30° C immediately following irradiation and processed for RNA extraction or stored at -80° C for future analysis. Cell survival rates were measured by plating recovered sham and irradiated cells on TGY plates for colony forming units (CFU) comparison.
|
| Growth protocol |
The strain Deinococcus radiodurans R1 (ATCC-13939) was grown overnight at 30° (D. radiodurans) in TGY broth (1% tryptone/0.1% glucose/0.5%yeast extract) to exponential phase (OD600 = 1).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted and resuspended in TRIzol reagent (Invitrogen, 15596-026), and lysed using a bead-beater (Bio Spec Products Inc, 3110BX) with four 100s pulses. The top aqueous phase containing RNA was extracted with chloroform:isoamyl (24:1) alcohol and precipitated with isopropanol. The resulting pellet was dissolved in RNAse-free water. RNA concentration was measured by spectrophotometer and stored in -20° C for short term use. The integrity and purity of total RNAs were verified with spectrometer (OD260/OD280) and RNA gel staining.
cDNA libraries were prepared from total RNAs that extracted from irradiated or non-irradiated cells. We used NEBNext® Small RNA Libray Prep Set for Illumina® (New England Biolabs Inc. E7330S) to prepare cDNA for all samples with the protocol provided by the manufacturer.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, then mapped to D. radiodurans whole genome using bowtie2.2.0
All intergenic loci mapped with over 100 reads per base and longer than 30 nt were annotated as potential sRNA candidates. Specific counts were calculated for 25 identified sRNAs.
Genome_build: ASM856v1; NC_001263.1 and NC_001264.1
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...
|
| |
|
| Submission date |
Jan 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Chen-Hsun Tsai |
| Organization name |
Univ. of Texas at Austin
|
| Department |
McKetta Department of Chemical Engineering
|
| Street address |
Room 3.416, 200 E. Dean Keeton St. Stop C0400
|
| City |
Austin |
| State/province |
Texas |
| ZIP/Postal code |
70712 |
| Country |
USA |
| |
|
| Platform ID |
GPL19650 |
| Series (1) |
| GSE64952 |
Transcriptional Analysis of Deinococcus radiodurans for Novel sRNAs expression that are Differentially Expressed under Ionizing Radiation |
|
| Relations |
| BioSample |
SAMN03283947 |
| SRA |
SRX841924 |
