|
| Status |
Public on Mar 09, 2024 |
| Title |
cmdT_pulldown |
| Sample type |
SRA |
| |
|
| Source name |
str. K12 substr. MG1655
|
| Organisms |
Escherichia coli; Escherichia phage T4 |
| Characteristics |
tissue: str. K12 substr. MG1655
treatment: T4 infection
|
| Growth protocol |
Cultures at an OD600 between 0.2 and 0.3 were infected with bacteriophage T4 at a multiplicity of infection (MOI) of 10 and continued to grow at 37C with shaking at 200rpm until sample collection. Samples were collected by mixing 1 mL of cells with 1 mL of of boiling lysis buffer (2% SDS, 4 mM EDTA) before flash freezing in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with two rounds of acidic phenol heated to 67C followed by one extraction with acidic phenol chloroform at room temperature. RNA was precipitated using isopropanol precipitation at -80C and DNA was removed with DNAse treatment using Turbo DNAse. Extracted RNA was ADP-ribosylated with CmdT with 0.25 mM 6-Biotin-17-NAD+ for 4 hours at 37 °C and then continued at 4 °C overnight. 10 µg of each reaction was kept at -80 °C as the pre-pulldown sample. The remaining 10 µg were incubated with streptavidin conjugated superparamagnetic beads following the manufacturer’s protocol. RNA was stripped from the beads by addition of 0.5 mL Trizol and incubation at 25 °C for 15 minutes on a thermomixer at 1000 rpm. Beads were then precipitated with a magnet and 100 µL of chloroform were added. The phases were separated by centrifugation at 14,000 g for 15 minutes. Finally, the aqueous phase was purified using the RNA Clean and Concentrate Kit (Zymogen).
cDNA library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following the manufacturer's protocol.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
DNBSEQ-G400 |
| |
|
| Description |
Singular G4 sequencer
Complete Genomics
|
| Data processing |
Remaining adapter sequences were trimmed from reads using cudadapt. Paired-end reads were mapped to the MG1655 (NC_00913.2) and T4 (NC_000866) genomes using bowtie2 and default settings. Mapped reads were converted into python operable numpy arrays using the python package genomearray3. TPM calculations were performed Geneious.
Assembly: NC_00913.2, NC_000866
Supplementary files format and content: TPM values for each gene in addition to the gene name are listed for both the MG1655 genome (ripseq_invitro_host_tpm.csv) and T4 genome (ripseq_invitro_t4_tpm.csv) with each column denoted a different sample.
|
| |
|
| Submission date |
Jan 17, 2024 |
| Last update date |
Mar 09, 2024 |
| Contact name |
Christopher Ross Doering |
| E-mail(s) |
cdoering@mit.edu
|
| Organization name |
MIT
|
| Department |
Biology
|
| Lab |
Michael Laub
|
| Street address |
31 Ames St
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL34101 |
| Series (2) |
| GSE253511 |
Anti-viral defense by an ADP-ribosyltransferase that targets mRNA to block translation (RIP-Seq in vitro) |
| GSE253514 |
Anti-viral defense by an ADP-ribosyltransferase that targets mRNA to block translation |
|
| Relations |
| BioSample |
SAMN39476096 |
| SRA |
SRX23265787 |
