|
| Status |
Public on Oct 06, 2020 |
| Title |
RNAseq_RRFKD_15min [ks21] |
| Sample type |
SRA |
| |
|
| Source name |
RNAseq_RRFKD_15min
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: RRFKD
rna source: whole cell lysate
growth condition: 37 C in arabinose, 37 in glucose for 10 min, 37 in glucose for 15 min
molecule subtype: mRNA
|
| Growth protocol |
Cells were cultured in LB+0.2% arabinose @ 37 C to OD = 0.2. The media was changed to LB+0.2% glucose and the cells were cultured at 37 C for 10 min. The media was changed one more time to fresh LB+0.2% glucose and the cells were cultured at 37 C and harvested after 5 min, 15 min, or 60 min by filtration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were cryogenically pulverized in a Spex 6870 freezer mill; 5 cycles of 1 min at 5 Hz with 1 min cooling. Cell lysates were clarified by centrifugation. For standard ribosome profiling (StRP), lysates were digested with nucleases, and monosomes were purified over a sucrose gradient. For high salt ribosome profiling (HighSaltRP), ribosomes were pelleted by sucrose cushion and then resuspended. Purified ribosomes were treated with nucleases, and monosomes were purified over a sucrose gradient.
For StRP and HighSaltRP, ribosome footprints were PAGE purified. Afterword, for all library types, RNA was ligated to a linker using RNA ligase T2, converted to DNA using RT, circularized, and PCR amplified.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
The linker sequence was trimmed and reads mapping to rRNA or tRNA sequences were discarded. The remaining reads were mapped to the E. coli K-12 MG1655 genome.
Genome_build: NC_000913.2
Supplementary_files_format_and_content: WIG files of ribosome occupancy, assigned to the 3'-end of reads, normalized by the total number of reads in the library.
|
| |
|
| Submission date |
Jun 03, 2020 |
| Last update date |
Oct 07, 2020 |
| Contact name |
Allen R Buskirk |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Street address |
725 N. Wolfe St
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL18956 |
| Series (1) |
| GSE151688 |
RRF plays critical roles in ribosome homeostasis in E. coli but has little effect on translational coupling |
|
| Relations |
| BioSample |
SAMN15091174 |
| SRA |
SRX8462276 |
