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| Status |
Public on May 05, 2019 |
| Title |
DMS-MaPseq on denatured E. coli total RNA (Replicate #1) |
| Sample type |
SRA |
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| Source name |
DH10B
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| Organism |
Escherichia coli |
| Characteristics |
strain: DH10B
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| Treatment protocol |
RNA was heated to 95 °C for 1 min in a total volume of 90 µl with 55 mM HEPES pH 7.9, 4.4 mM EDTA, 55% formamide. 10 µl of 0.5 M DMS in ethanol were then added to obtain a probing concentration of 50 mM. Probing was allowed to proceed at 95 °C for 1 min. Reactions were quenched by adding 200 µl RNA Binding Buffer (from RNA Clean & Concentrator-5 columns) supplemented with DTT to a final concentration of 750 mM. Samples were then purified on RNA Clean & Concentrator-5 columns.
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| Growth protocol |
LB medium, 37°C, 300 RPM
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets from exponential phase E. coli were resuspended in 1 ml of Resuspension Buffer [15 mM Tris-HCl pH 8.0; 450 mM Sucrose; 8 mM EDTA], and 18,500 U of Ready-Lyse Lysozyme (Epicentre, #R1810M) were added. After incubation at 22 °C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 x g (4 °C) for 5 min. Pellets were resuspended in 120 µl Protoplast Lysis Buffer [50 mM HEPES pH 8.0; 200 mM NaCl; 5 mM MgCl2; 1.5% SDS]. Samples were incubated for 5 min at 22 °C and for 5 min on ice, followed by addition of 30µl SDS precipitation buffer (50mM HEPES pH8, 1M KOAc, 5mM MgCl2). The precipitate was removed by centrifugation at 17000g (4 °C) for 5min and the supernatant was the extracted with TRIzol reagent, as per manufacturer instructions.
RNA was fragmented in 64 mM Tris-HCl pH 8.3, 96 mM KCl, 3.9 mM MgCl2 for 8 min at 94 °C and subsequently purified on RNA Clean & Concentrator-5 columns. Reverse transcription (RT) was performed using the TGIRT-III enzyme (InGex, #TGIRT50) as described previously (Zubradt et al., 2016). Briefly, to the 5 µl of fragmented RNA, 1 µl 10 mM dNTPs and 0.5 µl 10 µM random hexamers were added, and the mixture was heated to 70 °C for 5 min and immediately placed on ice for 1 min. 2 µl of 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], DTT to 5 mM, 10 U SUPERase In RNase Inhibitor, and 100 U TGIRT-III were added. Reverse transcription was then allowed to proceed at 25 °C for 5 min, followed by 1.5 h at 57 °C. To remove TGIRT-III from the RNA-DNA duplex, 1 µl of proteinase K was added to a final concentration of 0.1 µg/µl and the reaction incubated at 37 °C for 15 min., 1 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, #P8340) was then added to inhibit proteinase K activity. The reaction volume was then adjusted to 25 µl by adding 3 µl of 5X RT Buffer and 10 µl water. Libraries were then prepared using the TruSeq RNA Library Prep Kit v2 (Illumina, #15025063), starting from second strand synthesis.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
E. coli denatured (Replicate #1)
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| Data processing |
Library strategy: DMS-MaPseq
All the relevant analysis steps, from reads alignment to data normalization and structure modeling, were performed using the RNA Framework (Incarnato et al., 2018). Briefly, sequencing reads were clipped from adapter sequences and terminal positions with Phred qualities <20 were trimmed. Alignment was performed using the rf-map tool and the Bowtie v2 algorithm (Langmead et al., 2012), with soft-clipping enabled (parameters: -b2 -cp -b5 5 -mp "--very-sensitive-local"). Before proceeding to DMS-MaPseq data analysis, sequencing of an untreated IAV sample was performed in order to annotate eventual mutations with respect to the reference strain. Positions with mutations exceeding a frequency of 50% were annotated. All the subsequent analysis steps were then performed using this updated reference. Per-base mutation count and coverage were calculated using the rf-count tool (parameters: -nm -r -m -mq 0 -na -md 3 -ni). A ratiometric score was then calculated as ri = (mi / Ci) where ri, mi, and Ci are respectively the raw reactivity, the mutation count and the coverage at position i. Reactivity normalization was performed as a 2-step process. First, each sample was normalized by 90% Winsorizing, in order to smooth the contribution of both over and under-reactive residues, using the rf-norm tool (parameters: -sm 4 -nm 2 -rb AC). Briefly, each reactivity value above the 95th percentile was set to the 95th percentile and each reactivity value below the 5th percentile was set to the 5th percentile, then the reactivity at each position of the transcript was divided by the value of the 95th percentile. Reactivity data from targeted DMS-MaPseq analysis of probe pairing regions was independently Winsorized and then used to replace original RAPiD-MaPseq data. Second, both the in vitro and the in vivo datasets were normalized to the respective denatured controls as Ri = min(Si/Di,1) where Ri, Si, and Di are respectively the normalized reactivity, the sample reactivity (either in vivo or in vitro), and the denatured control reactivity at position i. This normalization yielded reactivity values comprised between 0 (low single-strandedness probability) and 1 (high single-strandedness probability). Given the high correlation of all the datasets, biological replicates were combined using the rf-combine utility following normalization. Combined datasets were then used for all downstream analyses.
Genome_build: A/Puerto Rico/8/1934(H1N1) from NC_002016 to NC_002023
Supplementary_files_format_and_content: For each DMS-MaPseq sample (but the untreated controls) a Wiggle track file is provided, reporting the mutation frequency of A/C residues for each Influenza A vmRNA. For the untreated controls, a multi-Fasta file is provided with the sequence of Influenza A vmRNAs, annotated with the detected SNVs. Additional processed files are available at: http://www.incarnatolab.com/datasets/IAV_Simon_2019.php
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| Submission date |
Nov 07, 2018 |
| Last update date |
May 06, 2019 |
| 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 |
GPL21222 |
| Series (1) |
| GSE122286 |
In vivo analysis of Influenza A mRNA secondary structures identifies critical regulatory motifs |
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| Relations |
| BioSample |
SAMN10392655 |
| SRA |
SRX4993246 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3463226_Ecoli_denatured_rep1.wig.gz |
22.6 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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