|
| Status |
Public on Mar 01, 2018 |
| Title |
QS+3 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells_QS+
|
| Organism |
Mesorhizobium ciceri biovar biserrulae WSM1271 |
| Characteristics |
plasmids: (pSDz-traR1)(pPR3-traI1)
od600nm: Late log phase
|
| Growth protocol |
Cultures were initially grown by streaking single colonies onto TY agar slopes and incubating for 5 days at 28°C. Two technical replicates of each slope culture was washed off into 50 mL TY broths and incubated for 24 h to achieve OD600 ~ 0.8. 12 mL of culturewas added to 24 mL RNA later solution (Qiagen) prior to RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellet was resuspended in 250 μL of 10 mM Tris-Cl (pH 8.0). This cell suspension was added to 2 mL lysis tubes filled with: 300 mg silica beads (0.1mm), 100 mg glass beads (0.1mm), 350 μL RLT buffer (Qiagen), and 3.5 μL β-mercaptoethanol and mechanically lysed in a FastPrep®-24 instrument (MP biomedicals) at speed 6.5 for 30 s. Total RNA was extracted from the lysate using a RNease Mini Kit (Qiagen) as per the manufacturers recommendations.
DNA was removed from approximately 3 μg of total RNA using the TURBO DNA-free™ kit (Invitrogen) and confirmed using a Qubit fluorometer dsDNA BR assay. rRNA was depleted from total RNA using a Ribo-Zero rRNA magnetic kit (Illumina) and resulting RNA was purified using a RNA Clean & Concentrator™ (Zymo Research). Barcoded cDNA libraries were prepared from rRNA depleted RNA samples using Ion Total RNA-Seq kit v2 (Thermo Fisher).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
processed data file:
DEseq2-NF
HTSeq-NF
DEseq2-F
HTSeq-F
|
| Data processing |
Read sets from technical repetitions were combined and adapter sequences were removed using nesoni clip (http://www.vicbioinformatics.com/software.nesoni.shtml). To reduce any potential rRNA/total-RNA abundance biases introduced during rRNA depletion, reads mapping to rRNA genes were removed using FastQ Screen (https://www.bioinformatics.babraham.ac.uk).
For differential gene expression analysis, read sets were filtered to remove sequences matching plasmids pPR3-traI1 and pSDz-traR1 prior to mapping with Bowtie2. An average (per biological replicate) of 14 million (standard deviation (SD) = 3.3 million) QS+ and 8.5 million (SD = 1.5 million) QS- post-filter reads were mapped to WSM1271 with 96.7-98.6% alignment rate. Differential gene expression analysis of the traI1 and traI2 untranslated mRNA promoter regions, PtraI1 and PtraI2, was carried out prior to filtering – as these reads were able to be distinguished from plasmid-borne mRNAs.
Read counts for gene features were performed using HTSeq with default settings then imported into DESeq2 for identification of differentially expressed genes using default parameters
Genome_build: NC_014923.1
Supplementary_files_format_and_content: HTSeq output (Microsoft excel worksheets) contains the raw HTSeq read counts for the RNASeq libraries QS-1a/b, QS-2a/b, QS-3a/b and QS+1a/b, QS+2a/b, QS+3a/b where reads corresponding to the plasmids pSDz-traR and pSDz-traI1 were filtered (HTSeq-F) or not filtered (HTSeq-NF) prior to mapping to the WSM1271 chromosome using Bowtie 2. DESeq2 files (Microsoft excel worksheets) contain DESeq2 output for differential gene expression analysis for each read count file (F or NF)
|
| |
|
| Submission date |
Jan 04, 2018 |
| Last update date |
Mar 01, 2018 |
| Contact name |
Timothy Haskett |
| E-mail(s) |
[email protected]
|
| Organization name |
Murdoch University
|
| Department |
Biological Sciences
|
| Lab |
Center for Rhizobium Studies
|
| Street address |
50 South Street Murdoch
|
| City |
Perth |
| State/province |
WA |
| ZIP/Postal code |
6150 |
| Country |
Australia |
| |
|
| Platform ID |
GPL24463 |
| Series (1) |
| GSE108732 |
RNASeq libraries of quorum-sensing induced and wild-type M. ciceri bv. biserrulae WSM1271 |
|
| Relations |
| BioSample |
SAMN08290852 |
| SRA |
SRX3530830 |
