|
| Status |
Public on Mar 29, 2016 |
| Title |
Rep2_MM_WT_Total mRNA |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells
|
| Organism |
Staphylococcus aureus |
| Characteristics |
growth phase: Exponentially growing S. aureus
media: MM
strain: USA300_FPR3757
genotype: wildtype
cdna type: total fragmented mRNA
|
| Treatment protocol |
To preserve ribosome occupancy on its mRNA template, 100 μg/ml of chloramphenicol were added to both WT and Δhpf cultures and incubated for 2 min before harvest.
|
| Growth protocol |
S. aureus cells (WT and Δhpf mutant) were grown in 1 liter of tryptic soy broth (TSB) or chemically defined minimal medium (MM) until OD600 ~0.4 at 37°C.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Bacterial cells were disrupted by cryomilling on a Retch MM400 (15 mm grinding balls, 10 ml jars) for 8 set of 3 min cycles at 15 Hz. Ref: Oh et al (2011) Cell 147: 1295-1308
Cell lysates were subjected to micrococcal nuclease digestion, and monosomes were collected by 10-55% sucrose density gradient. Total mRNA and the monosomal mRNAs were isolated by hot-phenol/chloroform method. Total mRNAs were fragmented by alkaline hydrolysis. The fragmented mRNAs and ribosome footprint mRNA (RPF) 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 |
| |
|
| 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 (CP000255.1) with Bowtie2.
The unaligned output files from Bowtie 2 (rRNA-less sequences) were mapped to the S. aureus USA300_FPR3757 (GeneBank CP000255.1) 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 custom scripts measured in RPKM (Rragments Per Kilobase of transcript per Million mapped reads)
Genome_build: GenBank CP000255.1
Supplementary_files_format_and_content: Tab-delimited text files include normalized read densities (RPM, reads per millions reads) at each nucleotide position
|
| |
|
| Submission date |
Oct 20, 2015 |
| Last update date |
May 15, 2019 |
| 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) |
| GSE74197 |
Ribosome hibernation factor promotes Staphyloccocal survival and differentially represses translation |
|
| Relations |
| BioSample |
SAMN04196691 |
| SRA |
SRX1357235 |
