|
| Status |
Public on Sep 19, 2016 |
| Title |
Spn_largeTranscr_t0min_caro_1 |
| Sample type |
SRA |
| |
|
| Source name |
planktonic cells
|
| Organism |
Streptococcus pneumoniae TIGR4 |
| Characteristics |
treatment: 0.25 µg/ml carolacton
optical density 600 nm: OD600 0.150
time point: 0min
|
| Treatment protocol |
At an OD600 of 0.15 treated cells were supplied with a final concentration of 0.25 µg/ml Carolacton in MeOH. Controls received the same volume MeOH. Six time-points (t) were chosen to be analyzed by RNA-seq. Prior to addition of Carolacton (t0), t5, t15, t60, t120, and t180 min after addition of Carolacton. At each point, 5 ml of culture were mixed with an equal volume RNAprotect (Qiagen), incubated at room temperature (RT) for 5 min, the cells harvested by centrifugation, and the pellet frozen and stored at -80°C.
|
| Growth protocol |
S. pneumoniae TIGR4 cells were inoculated to an optical density at 600 nm (OD600) of 0.1 and grown in Todd-Hewitt broth plus 1% yeast extract at 37°C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cellular RNAs were extracted using the miRNeasy kit (Qiagen) according to the manufacturers protocol for size-separation of RNA fractions >200 nt and </= 200 nt. Residual DNA in the fractions was digested by addition of 27 Kunitz units Dnase I (Qiagen), and absence of DNA verified by Polymerase chain reaction (PCR). mRNA enrichment was conducted for the samples containing RNAs >200 nt using the Ribo-Zero kit for Gram-positive bacteria (epicentre). RNA quality control of the RNA extracts, as well as the successful mRNA enrichment, was conducted by using an Agilent 2100 Bioanalyzer.
Libraries of RNA fractions >200 nt were constructed with the ScriptSeq V2 RNA-Seq Library Prep Kit (Illumina) according to the manufacturers instructions. Libraries of RNA isolations </= 200 nt were constructed with the TruSeq Small RNA Library Prep Kit (Illumina) also according to the manufacturers guidelines.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
mRNA-larger200nt_Rockhopper.txt
sRNA-larger200nt_HTSeq.txt
|
| Data processing |
Sequenced reads were trimmed for adaptor sequences using FastQC v0.11.2.
Raw read counts of mRNAs>200 nt were obtained by aligning samples 1-24 to the reference genome and counting mapped reads per feature using Rockhopper v2.0.3.
Raw read counts of tRNAs/mRNAs</=200 nt were obtained by aligning samples 25-36 to the reference genome and counting of the mapped reads per feature using Rockhopper v2.0.3.
Raw read counts of small regulatory RNAs>200 nt were obtained by first aligning samples 1-24 to the reference genome by using Bowtie2 v2.2.2 and then counting mapped reads at the location of previously selected small RNAs using HTSeq.
Raw read counts of small regulatory RNAs</=200 nt were obtained by aligning samples 25-36 to the reference genome by using Bowtie2 v2.2.2 and then counting mapped reads at the location of previously selected small RNAs using HTSeq.
Genome_build: NC_003028.3
Supplementary_files_format_and_content: The tab-delimited text files include raw read values for each time-point for both biol. replicates of control and carolacton-treated samples as obtained from Rockhopper (mRNAs/tRNAs) or HTSeq (sRNAs).
|
| |
|
| Submission date |
Jan 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jannik Donner |
| E-mail(s) |
[email protected]
|
| Organization name |
Helmholtz-Center for Infection Research
|
| Street address |
Inhoffenstr. 7
|
| City |
Braunschweig |
| ZIP/Postal code |
38124 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21349 |
| Series (1) |
| GSE76979 |
Differential transcription of small RNAs and mRNAs of Streptococcus pneumoniae TIGR4 when grown with the biofilm inhibitor Carolacton |
|
| Relations |
| BioSample |
SAMN04422056 |
| SRA |
SRX1535015 |
