|
| Status |
Public on Mar 09, 2017 |
| Title |
RF2*∆RF3_exp3_ribosome |
| Sample type |
SRA |
| |
|
| Source name |
whole cell lysate
|
| Organism |
Escherichia coli |
| Characteristics |
strain: K12 MG1655 prfB-Bstrain allele deltaprfC
|
| Treatment protocol |
Cells from each strain were rapidly harvested by filtration and lysate was produced by pulverization of liquid nitrogen cooled samples.
|
| Growth protocol |
All strains were grown shaking at 37°C in 200mL cultures of MOPS complete-glucose liquid media (unless otherwise annotated) in 1L flasks and cells were harvested at OD(420nm) between 0.4 - 0.6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole cell lysates were clarified by brief centrifugation. Ribosome footprints were created using Mnase digestion (45 enzyme units per absorbance unit of lysate at 260nm). Using sucrose gradients the monosome fraction of lysate was isolated and footprints were size selected and converted to a cDNA library. Total RNA was extracted from purified lysate for a simultaneous RNA-seq library production for total RNA samples. Small RNA and ribosomal RNA subtraction was performed in total RNA samples using MEGAClear (ThermoFisher) and MicrobExpress (ThermoFisher) kits. Subtracted total RNA was then subjected to alkaline fragmentation prior to library preparation.
A Linker-1 adapter (IDT) was ligated onto the 3' end of size selected RNA fragments, either ribosome footprints or fragmented total RNA. A cDNA library was created using Superscript III (ThermoFisher). After gel purification of the cDNA library all ssDNA fragments were circularized using CircLigase (Epicentre). For ribosome profiling samples rRNA containing circles were depleted using oligo-biotin and streptavidin coupled Dynabeads (ThermoFisher) subtraction. A PCR amplification off the circles using HF Phusion (NEB) completed the libraries by adding Illumina Tru-Seq adapters and a unique index for each sample.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ribosome protected RNA fragments
|
| Data processing |
Remaining linker-1 reads (CTGTAGGCACCATCAAT) were removed from fastq files using fastx_clipper
Reads were aligned to E.coli rRNA and tRNA using Bowtie v.0.12.0 allowing for one mismatch and reads aligning to more than one position in the genome were randomly assigned to one of those positions
The remaining reads were aligned to genome using Bowtie v.0.12.0 allowing for one mismatch and reads aligning in more than one position in the genome were discarded
The aligned reads were converted to BAM files and sorted and indexed using SAMtools
Wiggle files were created by center mapping the reads between 20-40bps. Using a 10bp trim on either side of the read then mapping the read across the remaining location using Plastid.
Genome_build: NC_000913
Supplementary_files_format_and_content: Bedgraph wiggle files were created for each sample and replicate for all reads aligned to the genome after computational rRNA and tRNA subtraction
|
| |
|
| Submission date |
Oct 13, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Natalie Baggett |
| E-mail(s) |
BAGGETT.NATALIE@GMAIL.COM
|
| Phone |
415-476-1493
|
| Organization name |
UCSF
|
| Department |
Microbiology and Immunology
|
| Lab |
Carol Gross
|
| Street address |
600 16th Street, S376
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL14548 |
| Series (1) |
| GSE88725 |
Global analysis of translation termination in E. coli using release factor manipulations |
|
| Relations |
| BioSample |
SAMN05907840 |
| SRA |
SRX2243610 |
