|
| Status |
Public on Nov 23, 2011 |
| Title |
dRNA-seq of Green barley plastids with TEX treatment |
| Sample type |
SRA |
| |
|
| Source name |
plastids of primary leaves from 11-day-old green albostrians seedlings
|
| Organism |
Hordeum vulgare |
| Characteristics |
strain: cv. "Haisa"; albostrians mutant
tissue: primary leaves of green seedlings
development stage: Mixed stage
treatment: TEX
|
| Growth protocol |
The barley mutant line albostrians was grown for 11 days in soil at 23 °C in a growth chamber with a photoperiod of 16h (light intensity: 150 mE s-1 m-2). The first leaves from completely green and white seedlings were used for plastid isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from green and white plastids was cleaned from genomic DNA contamination by gDNA Wipeout buffer (QIAGEN). For depletion of processed transcripts, 7 ug of RNA from each sample was treated with TerminatorTM 5’-phosphate-dependent exonuclease (TEX; Epicentre #TER51020) or in buffer alone for 60 min at 30 °C. 1 unit TEX was used per 1 ug total chloroplast RNA. Following organic extraction (25:24:1 v/v phenol/chloroform/isoamyalcohol), RNA was recovered by overnight precipitation with 2.5 volumes of ethanol/0.1M sodium acetate (pH 6.5). RNA was further treated with 1 unit TAP (tobacco acid pyrophosphatase; Epicentre, #T19100) for 1 hour at 37 °C to generate 5’-mono-phosphates for linker ligation, and again purified by organic extraction and precipitation as described above. After addition of specific 5'linkers (with unique tags for each library) and polyA-tailing, the RNA was converted into a cDNA library. The cDNA libraries were sequenced on a Roche FLX sequencer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
454 GS FLX |
| |
|
| Data processing |
For the mapping of the sequence reads, 5-linker and poly-A-tails clipped reads larger or equal to 18 nt were aligned to the barley chloroplast genome (NC_008590) using WU Blast 2.0 (http://blast.wustl.edu/) with the following parameters: -B=1 -V=1 -m=1 -n=-3 -Q=3 -R=3 -gspmax=1 -hspmax=1 -mformat=2 -e=0.0001. For each library, graphs representing the number of mapped reads per nucleotide were calculated as previously described (Sittka et al.,2008).
|
| |
|
| Submission date |
Nov 22, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
foerstner@zbmed.de
|
| Organization name |
ZB MED - Information Centre for Life Sciences
|
| Department |
Information Services
|
| Lab |
Förstner Lab
|
| Street address |
Gleueler Str. 60
|
| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
| |
|
| Platform ID |
GPL14919 |
| Series (1) |
| GSE33773 |
The primary transcriptome of barley (Hordeum vulgare L.) chloroplasts |
|
| Relations |
| SRA |
SRX109245 |
| BioSample |
SAMN00760752 |
