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Sample GSM6605713

Query DataSets for GSM6605713

Status

Public on May 25, 2023

Title

E. coli rS1-His, uninfected, R1 [LB_R1]

Sample type

SRA

Source name

Escherichia coli strain B with endogenously His-tagged rS1

Organism

Escherichia coli B

Characteristics

strain: Escherichia coli strain B with endogenously His-tagged rS1
medium: LB
growth stage: exponential
treatment: uninfected, LB added
spike-in: rS1-His RNAylated with RNAI
phage moi: no phage added
bacterial strain: Escherichia coli (Migula 1895) Castellani and Chalmers 1919 (DSM 613, ATCC 11303) modified with endogenously His-tagged rS1

Treatment protocol

T4 phage WT or T4 phage ModB R73A,G74A were added to an MOI of 5.0. For the uninfected negative control, same volumes of LB medium were added to the cultures. Cultures were then incubated at 37 °C for 8 min and E. coli harvested in by centrifugation at 3,000 x g for 13 min.

Growth protocol

100 ml cultures of E. coli B strain with endogenously His-tagged rS1 in LB supplied with 1 mM CaCl2, 1 mM MgCl2 and 30 µg/ml Kanamycin were grown at 37 °C in 250 ml baffled Erlenmeyer flasks to an OD600 ~ 0.8.

Extracted molecule

total RNA

Extraction protocol

Pellets from 100 ml culture infected with either T4 phage WT, T4 phage ModB R73A,G74A (R73A) or the uninfected control (LB) were resuspended in 2 ml Ni-NTA wash buffer (10 mM imidazole, 50 mM Tris-HCl pH 7.5, 1 M NaCl, 1 M urea, 5 mM MgSO4, 5 mM β-mercaptoethanol, 5 % glycerol, pH 8.0, EDTA-free protease inhibitor) on ice and lysed by sonication (6 min, 50 % amplitude, 0.5 s pulse). The lysate was cleared from cell debris by centrifugation at 21,000 x g, 4 °C, 30 min. 1.9 ml supernatant, 50 µl Ni-NTA agarose beads (Jena Bioscience, equilibrated in Ni-NTA wash buffer), 80 U murine RNase inhibitor and 4.72 µg of rS1 D2 RNAylated with NAD-capped RNAI were combined and incubated at 4 °C in a Rotary Mixer for 30 min. Entire samples were transferred to Mobicol mini spin columns. Beads were washed four times with 200 µl Ni-NTA wash buffer and subsequently eight times with 200 µl Strep wash buffer (50 mM Tris-HCl pH 7.5, 8 M urea). Beads were equilibrated in standard ligation buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.4) and blocked with BSA prior to 3’-RNA-adapter ligation at 4 °C overnight in the presence of standard ligation buffer, 50 mM β-mercaptoethanol, 0.05 µg/µl BSA, 15 % (v/v) DMSO, 5 µM adenylated RNA-3’-adapter, 0.5 U/µl T4 RNL1 (NEB) and 10 U/µl T4 RNL2, tr. K227Q (NEB). Protein was rebound by addition of NaCl to 1.5 M and incubation at 20 °C, 400 rpm for 20 min. Beads were subsequently washed six times with strep wash buffer and equilibrated in first strand buffer (50 mM Tris-HCl pH 8.3, 3 mM MgCl2, 75 mM KCl) and blocked with BSA. Reverse transcription of protein-bound RNA was performed in a 30 µl scale for 1 h at 40 °C using 10 U/µl Superscript IV Reverse Transcriptase (Invitrogen) in the presence of 5 µM RT primer, first strand buffer, 25 mM β-mercaptoethanol, 0.05 µg/µl BSA and 0.5 mM dNTPs. After incubation, NaCl was added to 1.5 M and incubated at 20 °C, 400 rpm for 1 h in order to rebind RNA-cDNA hybrids. Beads were subsequently washed five times with 0.25 x strep wash buffer (2 M urea, 50 mM Tris-HCl pH 7.5) and equilibrated in aExoI buffer (10 mM Tris-HCl pH 7.9, 5 mM β-mercaptoethanol, 10 mM MgCl2, 50 mM NaCl) and blocked with BSA. Residual RT primer was removed by ExoI digest with 1 U/µl E. coli ExoI (NEB) in ExoI buffer at 37 °C for at least 30 min. Beads were finally washed with 200 µl 0.25 x strep wash buffer five times and 200 µl immobilization buffer (10 mM Na-HEPES pH 7.2, 1 M NaCl) three times. cDNA was eluted by incubation of beads in 100 µl 150 mM NaOH at 55 °C for 25 min and by washing with 100 µl MQ water. Eluate pH was neutralized by addition of 0.05 volumes 3 M NaOAc pH 5.5. cDNA was removed from residual protein by phenol-chloroform extraction and precipitated with 2.5 volumes ethanol in the presence of 0.3 M NaOAc pH 5.5 overnight. Precipitated cDNA was C-tailed using 1 U/µl TdT (Thermo Fisher) in the presence of 1.25 mM CTP and 1 x TdT buffer at 37 °C for 30 min and subsequently inactivated at 70 °C for 10 min. 5 µM cDNA anchor (hybridization of fwd and rev anchor) were ligated to C-tailed cDNA in standard ligation buffer in the presence of 10 µM ATP and 1.5 U/µl T4 DNA Ligase (Thermo Fisher Scienttific) at 4 °C overnight. Ligation reactions were inactivated at 70 °C for 10 min and cDNA was ethanol-precipitated.
For the preparation of the Illumina RNAylome Seq library, cDNA was amplified by PCR using 2 U Phusion Polymerase (Thermo Fisher Scientific) in the presence of 5 % (v/v) DMSO, 200 µM dNTPs and 2500 nM NEBNext Universal and Index Primer each (Primer Set 1, NEB). PCR products were purified by native PAGE purification and ethanol-precipitated. dsDNA concentration was determined via Quantus fluorometer (Promega) and library size was determined with the Bioanalyzer (Agilent). Equimolar amounts of each library were sequenced on a MiniSeq system (Illumina) using the MiniSeq High-Output Kit (150 cycles, Illumina) generating 20 million 151 bp single-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiniSeq

Description

total RNA from E. coli cell lysate

Data processing

NGS data were demultiplexed using bcl2fastq (version 2.20.0, Illumina). Fastq files were assessed using FastQC (version 0.11.9) and Illumina sequencing adapters were trimmed from reads using cutadapt (version 1.18). Reads were aligned to a reference genomes composed of an E. coli K12 (U00096.3), bacteriophage T4 (NC000866.4) and RNAI (self-designed) with hisat2 (version 2.2.1). Primary alignments were selected using samtools (version 1.7) and reads per genomic feature were counted with featureCounts (version 2.0.1 from Subread package). The resulting counts table was subjected to further analysis and data visualisation in R (version 4.1.2). Read counts were normalized to the overall amount of mapped reads per sample and to the respective read counts for the RNAI spike-in.
Data visualisation was performed in R and alignments were manually inspected in Integrative Genomics Viewer (IGV, version 2.4.9).
Assembly: reference genomes composed of an E. coli K12 (U00096.3), bacteriophage T4 (NC000866.4) and RNAI (self-designed)
Supplementary files format and content: raw count data for all samples

Submission date

Sep 29, 2022

Last update date

May 25, 2023

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