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| Status |
Public on Sep 24, 2024 |
| Title |
DMS-MaPseq of E. coli TOP10, exponential phase, ex vivo, 37°C (total RNA) |
| Sample type |
SRA |
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| Source name |
TOP10
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| Organism |
Escherichia coli |
| Characteristics |
strain: TOP10
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| Treatment protocol |
For dimethyl sulfate (DMS; cat. D186309, Merck) probing, DMS from a fresh 1:4 dilution in ethanol (~2.64 M) was added to the bacteria at a final concentration of 200 mM. Probing was conducted for 2 min at 37°C, or for 30 min at 10°C (to achieve comparable modification efficiencies), with moderate shaking (800 RPM). Reactions were then quenched by addition of 1 volume 1 M DTT, after which bacteria were collected by centrifugation at 17,000g for 1 min. Supernatant was discarded, the pellet was washed twice with 0.5 M DTT, and then immediately subjected to RNA extraction.
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| Growth protocol |
E. coli K-12 MG1655 derivative strains DH5α and TOP10 (DH10b) were streaked on LB plates, and a single colony was picked, inoculated in 4 mL LB broth, and grown overnight at 37°C with shaking. The day after, the culture was diluted to an OD600 = 0.05 in 25 mL LB broth and grown at 37°C until OD600 ≈ 0.5 (~2 h). For cold-shock, 2 mL of this culture were mixed with 2 mL of LB broth pre-chilled to 0°C in a water-ice slurry, then incubated at 10°C for 20 min.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were resuspended in 62.5 μl Resuspension Buffer [20 mM Tris-HCl pH 8.0; 80 mM NaCl; 10 mM EDTA pH 8.0], supplemented with 100 μg/mL final Lysozyme (cat. L6876, Merck) and 20 U SUPERase•In™ RNase Inhibitor (cat. A2696, ThermoFisher Scientific), by vigorous vortexing. Samples were incubated at room temperature for 1 min, followed by addition of 62.5 μl Lysis Buffer [0.5% Tween-20; 0.4% Sodium deoxycholate; 2 M NaCl; 10 mM EDTA]. Samples were then inverted 5-10 times and incubated at room temperature for 2 min, followed by additional 2 min on ice. 1 mL ice-cold TRIzol™ Reagent (cat. 15596018, ThermoFisher Scientific) was then added, samples were vigorously vortexed for 15 sec, and RNA was extracted as per manufacturer instructions.
Prior to library preparation, highly abundant short RNA species, such as tRNAs, were depleted on Monarch® RNA Cleanup columns by loading a 1:1:1 mixture of total RNA in nuclease-free water, RNA binding buffer and 100% ethanol. rRNA depletion was performed on 1.1 μg total RNA using the RiboCop for Bacteria kit (cat. 126, Lexogen), with two minor changes to the manufacturer’s protocol: denaturation temperature was increased to 95°C, and probe annealing temperature was lowered to 55°C. Following rRNA depletion, RNA was cleaned up on Monarch® RNA Cleanup columns, eluted in 8 μl nuclease-free water, and supplemented with 2 μl 100 μM random hexamers, 2 μl dNTPs (10 mM each) and 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2]. Samples were then incubated at 94°C for 5.5 min, to simultaneously denature and fragment the RNA to a median size of 200 nt, and immediately transferred to ice for 1 min. Samples were then supplemented with 1 μl DTT 0.1 M, 20 U SUPERase•In™ RNase Inhibitor, 200 U TGIRT™-III Enzyme (InGex, cat. TGIRT50) and 25 ng/μl Actinomycin D (to increase strand-specificity; cat. A1410, Merck), and then incubated at 25°C for 10 min, 57°C for 1 h, and 60°C for 1 h. TGIRT-III was degraded by adding 2 μg Proteinase K and incubating at 37°C for 20 min. Proteinase K was inactivated by addition of Protease Inhibitor Cocktail (cat. P8340, Merck). cDNA-RNA hybrids were then converted to dsDNA using the NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module (cat. E7550, New England Biolabs), by incubating at 16°C for 1 h. DsDNA was then cleaned up with 1.8 volumes NucleoMag NGS Clean-up and Size Select beads (cat. 744970, Macherey Nagel), and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (cat. E7645S, New England Biolabs), as per manufacturer instructions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 1000 |
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| Data processing |
Following sequencing, paired-end reads were clipped of sequencing adapters using Cutadapt v4.4 (parameters: -A AGATCGGAAG -a AGATCGGAAG -m 100:100 -O 1), and merged using PEAR v0.9.11 (parameters: -n 100 -q 20 -u 0 -e -y 10G -z). Merged reads were then combined with R1 and the reverse-complemented R2 for read pairs that could not be merged. Next, a comprehensive annotation of E. coli transcriptional units with experimentally-determined TSSs was built by aggregating 5’ UTR information from RegulonDB and transcriptional units from EcoCyc (available from https://www.incarnatolab.com/datasets/EcoliEnsembles_Borovska_2024.php), and the corresponding sequences were extracted from the E. coli str. K-12 substr. MG1655 genome (GenBank: U00096.3). Reads were then mapped to this reference using the rf-map tool of the RNA Framework v2.8.3 and Bowtie2 v2.3.5.1, after clipping terminal bases with Phred quality < 20, discarding reads containing internal Ns, and trimming the 6 5’-most bases to account for possible mispriming artifacts (parameters: -cq5 20 -ctn -cmn 0 -cl 50 -mp "--very-sensitive-local --nofw" -b5 6). Alignments in SAM format were sorted and converted to BAM format using Samtools v1.15.1. BAM alignments were then processed using RNA Framework’s rf-count to generate both RC files (containing per-base mutations and coverage) and MM files (containing a map of mutated positions per read). Aligned reads spanning less than 100 nt of a transcript, as well as reads having more than 10% mutated bases, or less than 2 mutations, were discarded. Insertions and ambiguously aligned deletions, as well as deletions longer than 1 nt, were ignored. Mutations were considered only if both the mutated base and the two surrounding bases had Phred quality > 20, and consecutive mutations falling within 3 nt from each other were ignored (parameters: -m -mm -wl 2000 -ds 100 -es -na -ni -md 1 -dc 3 -me 0.1 -mpr 2). DMS-MaPseq data from total RNA, used for the calibration of folding parameters, was analyzed with minor changes to the above protocol. Briefly, reads were mapped to a reference composed only of the 16S and 23S rRNA sequences. The minimum length spanned by reads was decreased to 90 nt (as total RNA DMS-MaPseq experiments were sequenced as single-read 100 bp, in contrast to rRNA-depleted DMS-MaPseq experiments that were sequenced as paired-end 150 bp) and reads harboring < 2 mutations were also retained (parameters: -m -ds 90 -es -na -ni -dc 3 -ow -me 0.1 -md 1).
Assembly: U00096.3
Supplementary files format and content: Wiggle files of bulk raw DMS-MaPseq mutation frequencies
Library strategy: DMS-MaPseq
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| Submission date |
Nov 07, 2023 |
| Last update date |
Sep 24, 2024 |
| Contact name |
Danny Incarnato |
| E-mail(s) |
d.incarnato@rug.nl
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| Organization name |
University of Groningen
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| Department |
Molecular Genetics
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| Street address |
Nijenborgh 7
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| City |
Groningen |
| State/province |
Netherlands |
| ZIP/Postal code |
9747 AG |
| Country |
Netherlands |
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| Platform ID |
GPL33919 |
| Series (1) |
| GSE247244 |
Transcriptome-wide mapping of RNA secondary structure ensembles in a living cell |
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| Relations |
| BioSample |
SAMN38149817 |
| SRA |
SRX22411111 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7885845_TOP10_exvivo_rRNA.wig.gz |
23.3 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
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