|
| Status |
Public on Jun 01, 2021 |
| Title |
plus_toxIN_10min_rif_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial liquid culture
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
plasmid: pBR322-toxIN
time post-addition of rifampicin: 10
|
| Treatment protocol |
Cultures were back-diluted to OD600 = 0.2 and grown at 30 °C for 30 min before being treated with rifampicin at 300 μg/mL. At each timepoint post-treatment, 500 μL cells was mixed with 500 μL lysis buffer (SDS 2%, 4 mM EDTA pH = 8) and boiled at 100 °C for 5 min before being flash-frozen.
|
| Growth protocol |
Separate E. coli colonies were picked from LB agar plates for each replicate. Overnight cultures (12-16 hrs) were grown at 37°C in M9 media supplemented with 0.1% casamino acids, 0.4% glycerol, 0.4% glucose, 2 mM MgSO4, and 0.1 mM CaCl2. Overnight cultures were back diluted into fresh media and grown 3-4 hours to an OD600 of ~0.35 at 37°C in an orbital shaker at 200 RPM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using two rounds of hot acid-phenol extraction at 67°C, followed by one round of hot acid-phenol-chloroform extraction at 67°C, followed by isopropanol precipitation.
See publication for complete protocol. Briefly, samples were treated with bacterial Ribo-Zero kit (Illumina) or MicrobEXPRESS (ThermoFisher). Next, RNA was fragmented with RNA fragmentation reagents (Ambion). First strand cDNA synthesis was conducted using random primers and Superscript III (Invitrogen). To enable strand specificity, second strand synthesis was conducted using dUTP instead of dTTP with RNase H, E. coli DNA ligase, and E. coli DNA polymerase. Ends were repaired with T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. 3’ ends were adenylated with the Klenow fragment (3’>5’ exo-). Y-shaped adapters were ligated and 9-12 cycles of PCR were conducted with standard Illumina primers with multiplexing indexes. Libraries were extracted from an acrylamide gel and submitted for sequencing at the MIT BioMicro Center using standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Paired-end reads mapped to MG1655 genome using bowtie2 with default settings. Any adapter sequences were removed prior to mapping.
For each uniquely mapped fragment, at all aligned positions a count was added (fragment density mapping).
Counts were depth normalized to a pseudo-reference sample calculated from all samples (fragment density mapping).
Counts at all positions were log2 transformed and a cleavage ratio was calculated (+ toxIN: - toxIN) for assessing ToxN cleavage in E. coli transcripts
Genome_build: NCBI reference sequence: NC_000913.2 (E. coli)
Supplementary_files_format_and_content: rif_cleavageratio.csv.gz is a log-transformed list of sequencing depth-normalized counts for every genomic position at both strands for each sample as well as the average cleavage ratio for each timepoint calculated from both replicates.
|
| |
|
| Submission date |
Nov 18, 2020 |
| Last update date |
Jun 01, 2021 |
| Contact name |
Chantal K Guegler |
| E-mail(s) |
cguegler@mit.edu
|
| Phone |
6172533677
|
| Organization name |
MIT
|
| Department |
Biology
|
| Lab |
Laub
|
| Street address |
31 Ames Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL21117 |
| Series (2) |
| GSE161754 |
Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection [toxIN_riftreatment_RNAseq] |
| GSE161756 |
Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection |
|
| Relations |
| BioSample |
SAMN16828725 |
| SRA |
SRX9528615 |
