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| Status |
Public on Jul 03, 2020 |
| Title |
OX EE RNA Bio-replicate 1 |
| Sample type |
SRA |
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| Source name |
lab strain
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Characteristics |
strain: OX
growth phase: Early exponential
biological replicate number: 1
paired or single end: single
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| Treatment protocol |
Different phases of growth were harvested.
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| Growth protocol |
For performing growth experiments, overnight cultures were diluted to A600 of 0.003 in 25 ml LB in 250 ml conical flasks. These were incubated at 37 °C with 200 rpm orbital shaking.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At the EE or TS phases of growth, cells were harvested as described for RT-qPCR. The bacterial pellet was dissolved in TE pH 8.0 (100 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0) containing 0.5 mg/ml lysozyme. Lysis was carried out with 1% SDS and 0.1 mg/ml proteinase K while incubating at 40oC for 20 min. RNA was isolated by the addition of 0.3 M sodium acetate (pH 6.5) and 1 volume of phenol (pH 4.3):chloroform (1:1), followed by centrifugation (16000 x g, 4 °C, 10 min) in a phase lock tube (Quantbio, VWR) to separate the aqueous and organic phases. Following this, 1 volume of chloroform was added to the aqueous layer and centrifugation (16000 x g, 4 °C, 10 min) was repeated in the same phase lock tube. RNA was precipitated by the addition of 5 volumes of 100% ethanol and incubation at -20 oC for 1 h. RNA was pelleted by centrifugation (16000 x g, 4 °C, 10 min), washed once with 70% ethanol, and resuspended in 50 µl DEPC treated water. RNA was diluted to 500 ng/µl, denatured at 65oC for 5 min and treated with 10 U RNase-free DNase I (ThermoFisher Scientific, Waltham, USA) in DNase 1 buffer at 37 oC for 40 min. DNase treated RNA was cleaned up using the phenol chloroform method again.
Strand specific library preparation and sequencing of the DNase treated RNA was performed by Vertis Biotechnologie AG (Freising-Weihenstefan, Germany). Briefly, samples were analysed by capillary electrophoresis (Bioanalyzer, Agilent), and ribosomal RNA (rRNA) was depleted using the bacterial Ribo-Zero rRNA Removal Kit (Illumina). Ribodepleted RNA samples were fragmented using ultrasound (4 x 30 second pulses at 4oC). Oligonucleotide sequencing adapters were ligated to the 3’ end of each specific strand of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3’ end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis (Bioanalyzer, Agilent). For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200 - 550 bp using a differential clean-up with the Agencourt AMPure kit. An aliquot of the size fractionated pool was analyzed by capillary electrophoresis . Samples were PCR amplified for Truseq according to the instructions of Illumina. The cDNA pool was paired end sequenced on an Illumina NextSeq 500 system using 2 x 75 bp read length.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
rRNA depleted total RNA
MB_OX_fis_dps_EE_RNA_BR1
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| Data processing |
Alignment: bwa + samtools
Coverage calcuation (.bw files): deeptools
Counting reads mapping to genes: Rsubread
Calculating fold changes: EdgeR
Genome_build: NC_016810, NC_017718, NC_017719, NC_017720
Supplementary_files_format_and_content: raw_counts.xlsx: raw counts of reads mapping to genes; gene_fold_changes_with_FDR.xlsx: EdgeR calculated gene expression fold changes relative to WT; .bw: BigWig files for looking at coverage in a genome browser like IGB
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| Submission date |
Jun 10, 2020 |
| Last update date |
Jul 04, 2020 |
| Contact name |
Aalap Mogre |
| E-mail(s) |
aalap.mogre@gmail.com
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| Organization name |
Trinity College Dublin
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| Department |
Department of Microbiology
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| Lab |
Dorman Lab
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| Street address |
Moyne Institute of Preventitive Medicine
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| City |
Dublin |
| ZIP/Postal code |
D2 |
| Country |
Ireland |
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| Platform ID |
GPL20056 |
| Series (2) |
| GSE152227 |
Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes. [RNA-Seq] |
| GSE152228 |
Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes. |
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| Relations |
| BioSample |
SAMN15203249 |
| SRA |
SRX8524969 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4609635_MB_OX_fis_dps_EE_RNA_BR1_forward.bw |
1.4 Mb |
(ftp)(http) |
BW |
| GSM4609635_MB_OX_fis_dps_EE_RNA_BR1_reverse.bw |
1.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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