|
| Status |
Public on Jun 04, 2012 |
| Title |
DSH_PAO1_exp2 |
| Sample type |
SRA |
| |
|
| Source name |
Pseudomonas aeruginoasa PAO1 cDNA
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: PAO1
experiment: 2
size selection: 150-520 nt adapter ligated RNA
|
| Treatment protocol |
No additional treatments
|
| Growth protocol |
early stationary phase (A600 of about 2.6) cultures of P. aeruginosa strains PAO1 and PA14 grown at 37°C in 100 ml of Brain Heart Infusion (BHI) rich medium in 500-ml flasks vigorously shaken
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by RNeasy Kits (Qiagen) with modifications to enrich for small RNAs (<200 nt).
20 to 500 nt RNA was selected by PAGE and electroelution. RNA was first tagged at the 3’-end with 5'-monophosphate oligonucleotide adapters starting with three ribonucleotides followed by a sequence of 20 deoxyribonucleotides and terminally protected with an inverted dT.
RNase H-depletion of tRNA and 5S rRNA was performed essentially as described [PMCID 2665243] with some modifications. Size selection (40-520 nt for experiment 1(exp1) and 150-520 for experiment 2 (exp2)) were performed by PAGE/electroelution. cDNA was generated using the SMARTer™ PCR cDNA Synthesis Kit (Clontech).
Amplicon libraries were prepared for 454 pyrosequencing by PCR amplification with primers tailored for 454-sequencing and including Roche Multiplex Identifiers (MID) for “barcoding”. Free primers, were removed and final size selection (130 to 610 nt) performed by PAGE/electroelution.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
454 GS FLX Titanium |
| |
|
| Description |
small RNA profiling
|
| Data processing |
Adapters were trimmed using custom scripts written in Python. Reads were mapped to reference genomes [PAO1 genome build: NC_002516.1; PA14 genome build: NC_008463.1] using SEGEMEHL with default settings but reporting all equal best hits. WIG files prepared at single base resolution show log(coverage+1) for each genomic base on plus and minus strands of the reference genome sequences.
Genome Build:
DSH_PAO1_exp2_plus.wig: NC_002516.1
DSH_PAO1_exp2_minus.wig: NC_002516.1
|
| |
|
| Submission date |
Mar 07, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
David Stephen Horner |
| E-mail(s) |
david.horner@unimi.it
|
| Phone |
+39 02 503 14884
|
| Organization name |
Università degli Studi di Milano
|
| Department |
Molecular Biosciences and Biotechnology
|
| Lab |
Bioinformatics, Evolution and Comparative Genomics
|
| Street address |
Via Celoria 26
|
| City |
Milano |
| State/province |
MI |
| ZIP/Postal code |
20133 |
| Country |
Italy |
| |
|
| Platform ID |
GPL15322 |
| Series (1) |
| GSE36340 |
Comparative Profiling of Pseudomonas aeruginosa Strains Reveals Differential Expression of Novel Unique and Conserved Small RNAs |
|
| Relations |
| SRA |
SRX127960 |
| BioSample |
SAMN00809291 |
