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Sample GSM2516404

Query DataSets for GSM2516404

Status

Public on Jul 19, 2017

Title

DMS-seq (Ribo- RNA)

Sample type

SRA

Source name

DMS-seq (Ribo- RNA)

Organism

Escherichia coli

Characteristics

strain: K-12
substr: MG1655
molecule subtype: Total RNA (Cytosolic fraction)

Treatment protocol

A single colony of Escherichia coli was inoculated into 250 ml of LB medium without antibiotics, and grown at 37C with shaking (150 RPM) for approximately 4 hours, until OD600 was ~0.3 (log phase). EDTA was then added to a final concentration of 1 mM, and incubated for additional 5 minutes to make bacteria permeable to Actinomycin D. Actinomycin D was then added to a final concentration of 5 g/ml, and bacteria were incubated for additional 2 minutes to block transcription. 25 ml aliquots of bacteria were then rapidly pelleted by centrifugation at 4C. After centrifugation, medium was decanted, and cells from each 25ml of culture were resuspended in 1ml of structure probing buffer [10 mM HEPES-KOH pH 7.9; 140 mM NaCl; 3 mM KCl]. DMS was diluted 1:6 in 100% Ethanol to a final concentration of 1.76 M. Diluted DMS was added to bacteria to a final concentration of ~105 mM. Samples were incubated with moderate shaking (800 RPM) at 25C for 2 minutes, after which reactions were immediately transferred to ice. DTT was added to a final concentration of 0.7 M to quench DMS, and samples were vigorously vortexed for 10 seconds. Bacteria were then pelleted by centrifugation at 10,000g for 30 seconds (4C), and the supernatant was decanted. Pellets were then washed once with 1 ml Isoamyl alcohol (Sigma Aldrich, cat. W205702) to remove traces of water-insoluble DMS. Bacteria were then pelleted by additional centrifugation at 10,000g for 30 seconds (4C), supernatant was decanted, and samples were snap-frozen in liquid nitrogen. Pellets were stored at -80C.

Extracted molecule

total RNA

Extraction protocol

Each bacteria pellet from a 25 ml culture (OD600 ~0.3) was homogeneously resuspended in 200 µl of Buffer A [10 mM Tris pH 8.0; 20% Sucrose; 100 mM NaCl] supplemented with 200 U SUPERase• In™ RNase Inhibitor, by pipetting. 50 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0] supplemented with 1 µl Ready-Lyse™ Lysozyme Solution (Epicentre, cat. R1810M) were added dropwise, and the vial was gently tilted 5 times to ensure homogenous mixing. The sample was then incubated 1 minute at room temperature. 250 µl of Buffer C [0.5% Tween-20: 0.4% NaDOC; 2 M NaCl; 10 mM EDTA] were immediately added dropwise. The sample was then incubated 5 minutes at room temperature. At this stage the solution clears considerably without increasing its viscosity, and nucleoid becomes visible. Using a cut P1000 pipette tip, the whole sample was gently layered on the top of a 5-30% w/v sucrose gradient [10 mM Tris pH 8.0; 1 M NaCl; 1 mM EDTA; 1 mM DTT], and centrifuged at 17,000 RPM in a SW55Ti rotor (Beckman Coulter, cat. 342194) for 9 minutes (4°C). After centrifugation, the nucleoid fraction was collected using a syringe with a 18G blunt fill needle, and transferred to a new centrifuge tube. The remaining gradient was assumed to represent the cytosolic fraction. The nucleoid was then resuspended in 2.5 ml Wash & Resuspension buffer [40 mM Tris pH 7.5; 150 mM KCl; 10 mM MgCl2; 1 mM DTT; 0.01% Triton X-100] supplemented with 200 U SUPERase• In™ RNase Inhibitor, pulse vortexed for 5 seconds, and then centrifuged at 28,000 RPM in a SW55Ti rotor for 30 minutes (0°C). After centrifugation the supernatant was decanted, and the nucleoid pellet was washed twice with 2 ml of Wash & Resuspension buffer, taking care not to disturb it. The nucleoid was then resuspended in 500 µl Wash & Resuspension buffer, and solubilized by addition of 0.1 gr acid-washed glass beads (Sigma, cat. G1145), and shaking for 5 minutes in a TissueLyser (QIAGEN). For each 100 µl of purified nucleoids (or cytosolic fraction), 1 ml of TRIzol® Reagent (Invitrogen, cat. 15596-018) was added, and RNA was extracted following manufacturer’s instructions. RNA was analyzed on a 2100 Bioanalyzer (Agilent). In all experiments, RNA from cytosolic fraction (corresponding to mature RNA species) had RIN > 9.5. Total RNA yield from nucleoid fraction was ~6% of the total RNA content.
2 μg of RNA from the cytoplasmic fraction were diluted in 20 μl of Fragmentation buffer, and fragmented by incubation at 95°C for 5 minutes. Fragmented RNA was purified using RNA Clean & Concentrator™-5 columns. End repair of RNA fragments was performed in a final volume of 20 μl, in the presence of 20 U T4 Polynucleotide Kinase (NEB, cat. M0201L), and 20 U SUPERase• In™ RNase Inhibitor, by incubation at 37°C for 1 hour. End-repaired RNA was purified again using RNA Clean & Concentrator™-5 columns, and eluted in 6 μl of nuclease-free water. 6 μl of 2X RNA Loading Dye were added to end-repaired RNA. RNA was heated to 95°C for 2 minutes, and immediately placed on ice. Samples were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice corresponding to fragments above 200 nt was cut. The gel slice was crushed by centrifugation through a punctured 0.5 ml tube, and resuspended in 500 μl of Diffusion buffer supplemented with 60 U SUPERase• In™ RNase Inhibitor, then rotated at 4°C for 16 hours to allow passive diffusion of RNA fragments into buffer. RNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 6 μl nuclease-free water. 10 pmol of a pre-adenylated (rApp) adapter were ligated to size-selected RNA fragments in a reaction volume of 20 μl, using 400 U T4 RNA Ligase 2, Deletion Mutant in the presence of 20% PEG-8000, by incubation at 25°C for 2 hours. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and RNA was eluted in 5.5 μl nuclease-free water. RNA was heat-denatured at 70°C for 5 minutes, and reverse transcription was carried out in a final volume of 10 μl, in the presence of 0.5 mM dNTPs, 5 pmol of RT primer, 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, and 100 U SuperScript® III Reverse Transcriptase, by incubation at 50°C for 50 minutes. Template RNA was degraded by adding 1 μl of 1 M NaOH, and incubating at 95°C for 5 minutes. Reaction clean-up was performed using RNA Clean & Concentrator™-5 columns, and cDNA was eluted in 6 μl nuclease-free water. cDNA fragments were resolved on a 10% TBE-Urea polyacrylamide gel, and a gel slice corresponding to fragments in the range of 40-150 nt was cut (corresponding to truncated cDNA products). DNA was recovered by passive diffusion in Diffusion buffer for 16 hours at 37°C with moderate shaking. cDNA was precipitated by addition of 1 ml Isopropanol, and 2 μl Glycogen (20 μg/μl), and resuspended in 8.25 μl nuclease-free water. 10 pmol of a 5’-phosphorilated adapter were ligated to the 3’-OH of cDNA fragments in a final reaction volume of 25 μl, in the presence of 0.05 mM ATP, 20% PEG-4000, and 100 U CircLigase™ II ssDNA Ligase, by incubation at 60°C for 4 hours, and 68°C for 2 hours. Adapter-ligated cDNA fragments were purified from excess adapter using 1.8 volumes of Agencourt AMPure XP beads, following manufacturer’s instructions. cDNA was eluted in 20 μl of nuclease-free water, and indexed sequencing adapters were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext® High-Fidelity 2X PCR Master Mix.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

DMS-seq on cytosolic RNA fraction
processed data file: DMS-seq.rc.tar.gz

Data processing

library strategy: DMS-seq
FastQ files were examined using the FastQC tool. All the relevant SPET-seq data (nascent RNA) analysis and normalization steps were performed using a custom wrapper built on top of the RNA Framework13. Briefly, reads were clipped from 3’ adapter sequences using Cutadapt v1.10, discarding reads shorter than 15 nucleotides. Escherichia coli str. K-12 substr. MG1655 (GenBank: U00096.2) was used as the reference genome to extract transcripts’ sequences. Forward and reverse reads were independently mapped to the reference transcriptome using Bowtie v1.1.2, by allowing up to 7 mapping positions to enable mapping to the 7 E. coli rRNA genes (parameters: -n 2 -m 7 -a --best --strata -5 5 [--norc for forward reads, --nofw for reverse reads]). Forward and reverse mapped reads were then re-paired. Using reverse read mapping positions (corresponding to RNA Polymerase positions along gene), forward reads were split into separate SAM files for each transcription intermediate. When analysis was performed in deciles of transcription, genes were split into 10 equally sized deciles, and reads belonging to transcription intermediates falling in the same decile were pooled. SAM files were then passed to the rf-count tool of the RNA Framework to generate RT-stop counts (RC) files. Resulting RC files were normalized using the rf-norm tool of the RNA Framework in 50 nt sliding windows, with a 25 nt offset (parameters: -sm 2 -nm 2 -ec 0 -mc 0 -n 50 -nw 50 -wo 25). Mapping of DMS-seq data (mature RNA) was performed by using the rf-count tool (parameters: -cl 15 -bm 7 -ba -b5 5). Resulting RC files were normalized using the rf-norm tool (parameters: -sm 2 -nm 2 -ec 50 -mc 50 -n 50 -nw 50 -wo 25). The rf-norm tool generates a XML file for each transcript (or for each transcription intermediate/decile in the case of SPET-seq data). XML files for mature RNA were passed to the rf-fold tool of the RNA Framework (using ViennaRNA Package 2.2 with soft constraints14) to infer mature RNA structures (parameters: -md 600 -nlp).
Genome_build: U00096.2
Supplementary_files_format_and_content: Mapped reads were rescaled to a size of 10 bp, and BEDGraph files were generated using the genomeCoverageBed utility of the BEDTools suite.
Supplementary_files_format_and_content: DMS-seq data (pooled Total and Ribo- RNA) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data (pooled Total and Ribo- RNA from both replicates) is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. For each analyzed transcript, 10 files corresponding to SPET-seq data for the 10 transcription deciles are provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format
Supplementary_files_format_and_content: SPET-seq data is provided in the form of RNA Framework's (http://www.rnaframework.com) RNA Count (RC) files. A file corresponding to each transcription intermediate is provided. For a detailed description of the RC format, please refer to: http://rnaframework.readthedocs.io/en/latest/rf-count/#rc-rna-count-format

Submission date

Mar 01, 2017

Last update date

May 15, 2019

Contact name

Danny Incarnato

E-mail(s)

d.incarnato@rug.nl

Organization name

University of Groningen

Department

Molecular Genetics