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Sample GSM6447627

Query DataSets for GSM6447627

Status

Public on Nov 09, 2022

Title

E. coli, 20 min infected with T4 phage, R2 [t20_R2]

Sample type

SRA

Source name

Escherichia coli (Migula 1895) Castellani and Chalmers 1919 (DSM 613, ATCC 11303)

Organism

Escherichia coli B

Characteristics

medium: LB
growth stage: exponential
treatment: infected with T4 phage
time: 20 min
phage moi: 3.1
strain: Escherichia coli (Migula 1895) Castellani and Chalmers 1919 (DSM 613, ATCC 11303)
bacteriophage: Escherichia phage T4 (DSMZ, Braunschweig, Germany, DSM 4505)

Treatment protocol

Escherichia phage T4 (T4 phage) (DSMZ, Braunschweig, Germany, DSM 4505) suspension was added to a multiplicity of infection (MOI) of 3.1 to E. coli strain B in exponential growth phase and subsequently grown at 30 °C. 5 ml of culture were taken at 0 min (before infection), 1 min, 4 min, 7 min and 20 min post-infection and immediately lysed using the hot lysis method.

Growth protocol

A culture of E. coli strain B (DSMZ, Braunschweig, Germany, DSM 613) was grown at 37 °C in LB (Luria/Miller) medium supplemented with 1 mM CaCl2 and 1 mM MgCl2 to an OD600 of 0.5.

Extracted molecule

total RNA

Extraction protocol

Lysis and RNA extraction were performed with the hot lysis method. Therefore, 5 ml of sampled culture were incubated with 1 volume of lysis solution (1 % SDS, 4 mM EDTA) at 95 °C for 2 min each. 1 volume of water-saturated phenol (Roti-aqua phenol, Carl-Roth, Karlsruhe, Germany) was added to each sample and incubated at 67 °C for 10 min. Samples were centrifuged at 10,000 x g for 10 min, and the upper phase was added to 1 volume of phenol/chloroform/isoamyl alcohol (Carl-Roth, Karlsruhe, Germany). Samples were centrifuged again (10,000 x g, 10 min). The upper phase was precipitated by centrifugation (14,000 x g, 90 min, 4 °C) in the presence of 0.3 M sodium acetate and 1 volume isopropanol. The RNA pellet was resuspended in RNase-free water, and residual DNA was digested with 2 µl DNase I (Roche, Basel, Schweiz) in 1 x DNase buffer at 37 °C for 30 min. The RNA was extracted with 1 volume phenol/chloroform/isoamylalcohol (Carl-Roth, Karlsruhe, Germany) twice and residual phenol was removed by diethyl ether (Fisher Scientific, Schwerte, Germany) extraction. RNA was again precipitated in the presence of 0.3 M sodium acetate and 1 volume isopropanol by centrifugation (14,000 g, 4 °C, 90 min). The RNA pellet was resuspended in 50 µl RNase-free water each. The RNA concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, US) and a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, US). RNA integrity was evaluated on a Bioanalyzer (Agilent, Santa Clara, CA, US) using the RNA 6000 Nano Kit according to the manufacturer’s instructions.
1 μg total RNA was subjected to ribosomal RNA depletion (rRNA) by Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria) (Illumina, San Diego, CA, US). rRNA-depleted RNA was randomly sheared in 10 μL dH2O, at 94 °C 10 min. Fragmented RNA was processed using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, US) for Illumina. cDNA was barcoded by PCR amplification using Illumina TruSeq adapters for Illumina. Further cDNA size selection in the range of 300 to 500 bp was performed, employing the Agencourt RNAClean XP kit (Beckman Coulter, Brea, CA, US). Primer-depleted cDNA was examined by Bioanalyzer and the concentration was measured by Qubit(Thermo Fisher Scientific, Waltham, MA, US). Multiplexed libraries were sequenced on a NextSeq 500 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

total RNA with depletion of rRNA

Data processing

The quality of reads obtained from Illumina RNA-Seq was assessed pre- and post-adapter trimming using FastQC (version 0.11.9). Fastq files were processed using the cutadapt tool (version 1.18) in order to remove reads containing Illumina TruSeq adapter sequences. Reads were aligned to the genome of E. coli (U00096.3) and bacteriophage T4 (NC_000866.4) using the hisat2 aligner (version 2.2.1) at default settings. Thereby, 88.71 to 96.51 % of reads were successfully aligned to the reference genomes. Samtools (version 1.7) was applied to select for primary alignments. Bam files were manually inspected as genomic maps using the Integrative Genomics Viewer (version 2.4.9). The reads mapped to individual features (annotated in gff3 files for U00096.3 and NC_000866.4 as gene) were quantified using featureCounts (Subread package version 2.0.1) with default settings while excluding reads overlapping multiple features. Using R (R version 4.1.2), genes annotated as rRNA (22 genes) from E. coli, which account for up to 0.6 % of reads per sample (Supplementary Fig. 2), were manually removed from the counts table prior to further analysis because they have been depleted from the total RNA before sequencing. The count data was normalized to transcripts per million (TPM) which allows to compare expression levels of genes between samples. Sample clustering was assessed by PCA with the prcomp package. The fractions of TPM normalized reads per sample and entity (T4 phage or E. coli) were calculated accordingly. Genes with low read counts (average read count below 1.5 across all samples) were removed from the TPM normalized count data including 338 E. coli and 17 T4 phage genes. These genes would otherwise confuse the analysis due to low and variable read counts. Further analyses in R focussed on data visualization using the pheatmap (1.0.12) and ggplot2 (3.3.6) package. Differential gene expression analysis during the early infection phase was assessed with DESeq2 applying a Wald-test at a log2fold change greater or smaller than 0 with a Benjamini-Hochberg-corrected p-value threshold of 0.05. Other infection phases were not assessed by DESeq2 as dramatic changes in the host and phage transcriptome have already been recorded 4 minutes post infection. Assignment of differentially expressed genes to Clusters of Orthologous Groups (COGs) was conducted in R using the COG database from the National Center for Biotechnology Information.
Assembly: E. coli (U00096.3) and bacteriophage T4 (NC_000866.4)
Supplementary files format and content: one table is provided which contains TPM normalized count data for all samples substracted by rRNAs

Submission date

Aug 11, 2022

Last update date

Nov 09, 2022

Contact name

Katharina Höfer

E-mail(s)

Katharina.Hoefer@synmikro.mpi-marburg.mpg.de

Organization name

Max-Planck-Institute for terrestrial Microbiology

Street address

Karl-von-Frisch-Str. 14