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| Status |
Public on May 04, 2022 |
| Title |
USA300_CcpA-HTF_biolrep1_techrep1 |
| Sample type |
SRA |
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| Source name |
CcpA-HTF cells
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| Organism |
Staphylococcus aureus subsp. aureus USA300 |
| Characteristics |
strain: USA300 LAC
genotype: CcpA-HTF
treatment: Grown to OD600 ~3.0 in TSB, shifted to Low Phosphate Medium (LPM) and UV cross-linked in the Vari-X-linker at 1000mJ/cm2. Harversed on filter paper by vacuum filtration and stored at -80ºC
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| Treatment protocol |
Cells were subsequently UV cross-linked (254 nm; 1000 mJ/cm2) in the Vari-X-linker (UVO3; (McKellar et al., 2020; Van Nues et al., 2017)), collected by vacuum filtration and flash-frozen on the filters and stored at -80ºC. As negative controls we either used UV irradiated cells from the USA300 parental strain.
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| Growth protocol |
S. aureus cells expressing HTF-tagged proteins were grown to saturation in TSB at 37 °C. Cells were then diluted in 100 ml fresh TSB to OD600 0.05, grown to an OD600 of roughly 3. Subsequently, cells were harvested by vacuum filtration and shifted to an equivalent volume of Low Phosphate Medium (LPM) for 15 minutes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the CRAC experiments, the cells were washed off the filters with 25ml of ice-cold Phosphate Buffer Saline (PBS) and pelleted by centrifugation. Cells (up to 0.5 grams) were subsequently resuspended in 2 volumes/cel weight of TN150-Lysostaphin buffer (50 mM Tris pH 7.8, 150 mM NaCl, 100 µg/mL Lysostaphin, 0.1% NP-40, 0.5% Triton X-100) and transferred to 5 mL screw-cap tubes (eppendofr). Sixty µL of RQ DNase 1 and 10 µL of SUPERase·In was added to the mixture and incubated at 37°C for 10 min to lyse the cells. 3V/cell weight of Zirconia beads (Thistle Scientific; 0.1 µm) were added and the mixture was vortexed vigorously 5 times for one minute with one-minute incubations on ice between each step. 2V/cell weight of cold TN150 anti-peptidase (50 mM Tris pH 7.8, 150 mM NaCl, 1 Roche EDTA-free mini pellet, 0.1% NP-40, 0.5% Triton X-100 and 10 mM EDTA) buffer before centrifugation for 30 minutes at 10000 g at 4°C. Subsequently, 75µl Anti-FLAG® M2 Magnetic Beads (pre-washed with 3 times 1 mL TN150 buffer) was added and incubated with the lysate for 2 hours at 4ºC. The beads were then washed three times five minutes with 2 mL TN1000 buffer (50 mM Tris pH 7.8, 1M NaCl, 0.1% NP-40, 0.5% Triton X-100) and three times with 2 mL TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40, 0.5% Triton X-100) for 5 minutes. Beads were subsequently resuspended in 250 µl of TN15, 10 µL of home-made GST-TEV protease was added and the samples were rotated for 1.5-2 hours at room temperature. TN150 was added to a final volume of 600 µL and 550 µL of the TEV eluate was incubated with RNace-it™ (Agilent Technologies1 µL of 1:100 dilution) for exactly five minutes at 37ºC. Subsequently, 500 µl of the mixture was added to a tube with 0.4 g Guanidium-HCl and vortexed vigorously to inactivate the RNases. NaCl and imidazole (pH 8.0) were added to a final concentration of 300 mM and 10 mM, respectively and the mixture was transferred to 50µl of Ni-NTA agarose beads (QIAGEN) equilibrated with Wash buffer I (300 mM NaCl, 10 mM imidazole, 6M GuHCl, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM β- mercaptoethanol (βMe) and 0.5% Triton X-100) and incubated at 4ºC overnight on a rotator. The next day the beads were transferred to Pierce snap-cap columns (Thermo Scientific) and washed twice with 500 µL Wash buffer I and three times with 500 µL 1x NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% NP40, 5 mM βMe and 0.5% Triton X-100).
Radioactive labelling of 5’ ends, linker ligation and cDNA library preparation was performed as described previously (McKellar et al., 2020).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Cross-linked RNAs from CRAC on the USA300 CcpA-HTF strain, first biological replicate, first technical replicate
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| Data processing |
Library strategy: CLIP-seq/CRAC
Two biological CcpA CRAC experiments were performed, each with three technical replicates. The cDNA libraries were sequenced on Illumina NovoSeq6000 machines (NovoGene) and Illumina MiniSeq systems. Raw sequencing reads in fastq files were processed using the paired-end CRAC data analysis pipeline developed by Sander Granneman, which uses tools from the pyCRAC package (version 1.5.0) (Webb et al., 2014). The CRAC_pipeline_PE.py script first demultiplexes the data using pyBarcodeFilter.py and the in-read barcode sequences found in the L5 5’ adapters. Flexbar then trims the reads to remove 3’-adapter sequences and poor-quality nucleotides (Phred score <23). Using the random nucleotide information present in the L5 5’ adaptor sequences, the reads are then collapsed to remove potential PCR duplicates using pyFastqDuplicateRemover.py. The reads were then mapped to the JKD6008 and USA300 genomes using Novoalign (www.novocraft.com).
To determine to which genes the reads mapped to, we generated an 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 (Tjaden, 2015) on USA300 rRNA-depleted total RNA-seq data (Stuart McKellar and Ivayla Ivanova, unpublished) 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. Overlapping features, such as sRNAs that overlap with UTRs of protein-coding genes, were removed from the GTF file.
We then used pyReadCounters.py with Novoalign .novo or .sam output files as input and the GTF annotation file to count the total number of unique cDNAs that mapped to each gene. Reads that mapped to multiple features in the genome were randomly distributed over the features.
The individual tables generated by pyReadCounters were subsequently merged into a single table (WT_and_CcpA_raw_counts_rep1.txt and WT_and_CcpA_raw_counts_rep2.txt).
Because the technical replicates indicated high reproducibility, the raw counts of the technical replicates from the CcpA-HTF samples were subsequently merged to generate total coutns fro the two biological replicates. The negative control data were not further analysed as the counts for the total number of mapped reads for the parental control samples were extremely low compared to the CcpA-HTF data.
Genome_build: Staphylococcus_aureus_usa300_FPR3757
Supplementary_files_format_and_content: text, bedgraph, fasta and GTF files
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| Submission date |
Dec 22, 2020 |
| Last update date |
May 04, 2022 |
| Contact name |
Sander Granneman |
| E-mail(s) |
Sander.Granneman@ed.ac.uk
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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 |
GPL29525 |
| Series (1) |
| GSE163719 |
Global mapping of RNA-binding domains in multi-drug resistant Staphylococcus aureus |
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| Relations |
| BioSample |
SAMN17138537 |
| SRA |
SRX9719082 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4984365_CcpA_biolrep_1_techrep_1_minus_strand_reads.bedgraph.gz |
74.6 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM4984365_CcpA_biolrep_1_techrep_1_plus_strand_reads.bedgraph.gz |
45.8 Kb |
(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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