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Status |
Public on Aug 16, 2018 |
Title |
4_h_MMC_fragmented |
Sample type |
SRA |
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Source name |
4_h_MMC_total bacterial RNA
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Organism |
Caulobacter vibrioides NA1000 |
Characteristics |
genotype/variation: wild type internal stock id: KFS_0006 treated with: 0.1 ug/mL mitomycinC (MMC) for 4hrs
|
Treatment protocol |
Samples were collected prior to treatment (OD660 of 0.4) and after 4h of treatment with mitomycinC (0.1 ug/mL)
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Growth protocol |
Caulobacter crescentus was grown aerobically at 30C in PYE medium
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted with the HotPhenol method and digested with DNaseI For the depletion of processed transcripts, equal amounts of RNA were incubated with Terminator 5'-phosphate-dependent exonuclease (TEX) (Epicentre #TER51020) as previously described (Sharma et al., 2010). The transcripts were not fragmented in order to get mainly sequencing reads of the 5'-end of the transcripts. Libraries for Illumina sequencing of cDNA were constructed by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/), as described previously for eukaryotic microRNAs (Berezikov et al., 2006) but omitting the RNA size-fractionation step prior to cDNA synthesis. Equal amounts of RNA samples were poly(A)-tailed using poly(A) polymerase. Then, the 5'-triphosphates were removed by applying tobacco acid pyrophosphatase (TAP) resulting in 5'-monophosphat. Afterwards, a RNA adapter was ligated to the 5'-phosphate of the RNA. First-strand cDNA was synthesized by an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. In a PCR-based amplification step using a high fidelity DNA polymerase the cDNA concentration was increased to 20-30 ng/µl. A library-specific barcode for multiplex sequencing was part of a 3'-sequencing adapter.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
Quality trimming with fastq_quality_trimmer (fastx 0.11.2) Read mapping with READemption (0.4.3) and segemehl (0.2.) Coverage plote generation with READemtpoin Genome_build: NC_011916.1 Supplementary_files_format_and_content: wiggle of normalized coverages
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Submission date |
Sep 25, 2017 |
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 |
GPL21317 |
Series (1) |
GSE104186 |
Identification of small RNAs expressed in Caulobacter crescentus in response to DNA damage |
|
Relations |
BioSample |
SAMN07692733 |
SRA |
SRX3210365 |