|
| Status |
Public on Jan 16, 2024 |
| Title |
YP2_RPF_KES34_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
YP2_RPF_KES34_bacterial cells
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: USA300_FPR3757
genotype/variation: KES34 (unmodified ribosomes)
growth phase: Exponentially grown
cdna type: Ribosome-protected mRNA fragments (RPFs)
|
| Treatment protocol |
Unlike other Ribo-seq protocols, translation inhibitor was not added to the culture.To preserve ribosome occupancy on its mRNA template, cells were rapidly collected via vacuum filtration through 0.45 µm nitrocelluloase membrane.
|
| Growth protocol |
S. aureus cells were grown in 1 liter of tryptic soy broth until mid-log phase (OD600 ~0.8-1.0) at 37°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial cells were disrupted by cryomilling on a Retch MM400 (15 mm grinding balls, 10 ml jars) for 6 set of 3 min cycles at 15 Hz. Ref: Oh et al (2011) Cell 147: 1295-1308
Cell lysates were subjected to micrococcal S7 nuclease digestion, and 70S monosomes were collected by 10-40% sucrose density gradient. Total mRNA (alkaline hydrolysis) and the monosomal mRNAs (26-42 nt) were isolated by hot-phenol/chloroform method.
The fragmented mRNAs and ribosome footprint mRNAs (RPFs) were ligated to a universal linker. Following rRNA subtraction, PAGE purification, reverse transcription, cDNA circularization, and multiplexing PCR amplification, the cDNA libraries were sequenced on Illumina HiSeq 2000
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
YP-2_S2_L006
|
| Data processing |
CASAVA-1.8.2 basecalling
Raw FastQ sequencing data were processed on local instance of Galaxy platform.. Sequence libraries were pre-processed by clipping the adaptor sequence (5’CTGTAGGCACCATCAAT3’) and trimming the sequences by discarding the first base and all those extending beyond the 50th base. The sequencing reads were mapped to the rRNA reference genome with Bowtie2.
The unaligned output files from Bowtie 2 (rRNA-less sequences) were mapped to the S. aureus NCTC 8325 (GeneBank CP000253) with Bowtie v.0.12.0. The alignment .map files were used as inputs for the modified Python scripts to obtain normalized read density in "reads per million mapped reads" (RPM) at each nucleotide positions. Original Python scripts have been published in Ref: Becker et al (2013)Nature Protoc 8: 2212-2239
mRNA abundance were determined by Cufflinks in FPKM (Fragments Per Kilobase of transcript per Million mapped reads)
Differential gene expression was measured by Cuffdiff. Geometric library normalization and pooled dispersion estimation methods were employed with a 0.05 false discovery rate and a minimum alignment count of 10.
Genome_build: GenBank: CP000255.1
Supplementary_files_format_and_content: Tab-delimited text files include normalized-read densities (RPM) at each nucleotide position
|
| |
|
| Submission date |
Mar 04, 2021 |
| Last update date |
Jan 16, 2024 |
| Contact name |
Mee-Ngan F. Yap |
| E-mail(s) |
frances.yap@northwestern.edu
|
| Phone |
312-503-3793
|
| Organization name |
Northwestern University Feinberg School of Medicine
|
| Department |
Microbiology-Immunology
|
| Lab |
Searle 3-430
|
| Street address |
320 E Superior St
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL17452 |
| Series (1) |
| GSE168265 |
Comparative Ribo-Seq of Staphylococcus aureus harboring A2058-unmodified ribosomes and m6A2058-modified ribosomes |
|
| Relations |
| BioSample |
SAMN18140848 |
| SRA |
SRX10239935 |
