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| Status |
Public on May 04, 2022 |
| Title |
WT_rep1 |
| Sample type |
SRA |
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| Source name |
cells
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| Organism |
Staphylococcus aureus |
| Characteristics |
strain: USA300 LAC pCN33
treatment: Grown to OD600 ~3.0 in TSB + Erythromicin , harversed by centrifugation and stored at -80ºC
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| Growth protocol |
S. aureus cells were grown to saturation in TSB at 37 °C. Cells were then diluted in 20 ml fresh TSB supplemented with erytrhomicin to OD600 0.05 and then grown to an OD600 = 3. Subsequently, cells were harvested by centrifugation, and the cell pellets were snap frozen and stored at -80C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of RNA from S. aureus was performed as previously described4. Briefly, cells were cultured to the desired optical density and rapidly harvested by centrifugation at 8,600 g for 5 min, 4°C. Cell pellets were incubated with 100 µl of guanidium thiocyanate (GTC)-Phenol mix (pH 5.4; 1:1 ratio) and lysed by vortexing the cells for 5 minutes with 100 µl of Zirconia beads (0.1 mm; Biospec products 11079101z). Subsequently, 550 µl of GTC was added and the mixture was vortexed for several minutes. After a 10-minute incubation at 65ºC for 10 minutes, the mixture was cooled on ice for 10 minutes. Phases were separated by adding 300 µl chloroform isoamylalcohol (24:1) and by adding 1/10th of a volume of 3 M NaAc (pH 5.2) followed by vigorous vortexing. After 5 minutes of centrifugation in an Eppendorf centrifuge (12,400 g), the RNA was purified from the upper phase via two additional rounds of phenol-chloroform extraction and precipitated with 3 volumes of 96% cold ethanol. Pellets were washed with 1ml of 70% cold ethanol, air dried and resuspended in DEPC-treated water.
RNA from three experimental replicates was extracted as described above and sequenced on an Illumina Novoseq6000 machine using the TruSeq library preparation protocol (Novogene).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Total RNA of parental strain USA300, first biological replicate. Cells contained the empty pCN33 vector.
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| Data processing |
Three experimental replicate datasets from the USA300 parental strain and USA300 DccpA processed using Flexbar to remove poor quality nucleotides (Phred score <23) and adapter sequences. The reads are then mapped to the USA300 genome using Novoalign (version 2.07). Reads that mapped to multiple features in the genome were randomly distributed over the features. To determine to which genes the reads mapped to, we generated a modified USA300 annotation file in the Gene Transfer Format (GTF). This file contains the start and end positions of each gene on the chromosome as well as what genomic features (i.e., sRNA, protein- coding, tRNA) it belongs to. To generate this file, we used the Rockhopper software on all our USA300 rRNA-depleted total RNA-seq data and a minimal GTF file obtained from ENSEMBL (without UTR information). The resulting GTF file contained information not only on the coding sequences, but also more complete 5’ and 3’ UTR coordinates. We then used pyReadCounters from the pyCRAC package (version 1.5.1) to count the total number of reads that map to each gene and genomic features. These tables with raw counts were subsequently used to perform a differential expression analyses using DESeq2.
The pyGTF2bedGraph script was used to convert the data to bedgraph files. The data in the bedgraph files has been normalised to 'read per million' (rpm).
Genome_build: USA300_FPR3757
Supplementary_files_format_and_content: text, bedgraph, fasta and GTF files
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| Submission date |
Dec 02, 2021 |
| Last update date |
May 04, 2022 |
| Contact name |
Sander Granneman |
| E-mail(s) |
[email protected]
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| Organization name |
University of Edinburgh
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| Department |
Centre for Synthetic and Systems Biology
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| Lab |
Granneman lab
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| Street address |
Mayfield Road, Kings Buildings, Waddington building, room 3.06
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JD |
| Country |
United Kingdom |
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| Platform ID |
GPL27158 |
| Series (1) |
| GSE189977 |
Global identification of RNA-binding proteins and RNA-binding domains in methicillin-resistant Staphylococcus aureus |
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| Relations |
| BioSample |
SAMN23563347 |
| SRA |
SRX13278969 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5710779_WTrep1_1_hittable_reads.txt.gz |
40.0 Kb |
(ftp)(http) |
TXT |
| GSM5710779_WTrep1_1_minus_strand_reads.bedgraph.gz |
4.8 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5710779_WTrep1_1_plus_strand_reads.bedgraph.gz |
5.1 Mb |
(ftp)(http) |
BEDGRAPH |
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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