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Sample GSM4914588 Query DataSets for GSM4914588
Status Public on Jun 01, 2021
Title minus_toxIN_10min_T7_rep1
Sample type SRA
 
Source name Bacterial liquid culture
Organism Escherichia coli str. K-12 substr. MG1655
Characteristics strain: MG1655
phage: T7
plasmid: pBR322 empty vector
time post-infection: 10
ercc standard: Prior to extraction
Treatment protocol Cultures were back-diluted to OD600 = 0.2 and grown at 30 °C for 30 min before being infected with T7 at MOI = 5. At each timepoint post-infection, 500 μL infected 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 a custom rRNA depletion kit. 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 and T7 genomes 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).
For each uniquely mapped fragment, a count was added to the middle of each fragment (middle fragment mapping).
Counts were depth normalized to a pseudo-reference sample calculated from all samples (fragment density mapping) or by rpkm in each individual sample (middle fragment mapping).
Counts at all positions were log2 transformed and a cleavage ratio was calculated (+ toxIN: - toxIN) for assessing ToxN cleavage in transcripts; this analysis was conducted separately for the E. coli and T7 transcriptomes.
Genome_build: NCBI reference sequence: NC_000913.2 (E. coli); V01146.1 (T4)
Supplementary_files_format_and_content: t7_phage_cleavageratio.csv.gz is a log-transformed list of sequencing depth-normalized counts for the T7 genome for every genomic position at both strands for each sample as well as the average cleavage ratio for each timepoint calculated from both replicates.
Supplementary_files_format_and_content: t7_host_cleavageratio.csv.gz is a log-transformed list of sequencing depth-normalized counts for the E. coli genome for every genomic position at both strands for each sample as well as the average cleavage ratio for each timepoint calculated from both replicates.
Supplementary_files_format_and_content: t7_ERCCspikein_sum.csv.gz is the list of the sum of raw counts for the ERCC spike-in controls for each sample.
Supplementary_files_format_and_content: t7_phage_rpkm.csv.gz is the list of the rpkm per T7 mRNA for every sample at every timepoint calculated from both replicates.
Supplementary_files_format_and_content: t7_host_rpkm.csv.gz is the list of the rpkm per E. coli CDS for every sample at every timepoint calculated from both replicates.
 
Submission date Nov 19, 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)
GSE161756 Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection
GSE161795 Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection [toxIN_T7infection_RNAseq]
Relations
BioSample SAMN16834505
SRA SRX9531504

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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