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| Status |
Public on May 05, 2019 |
| Title |
DMS-MaPseq on in vivo spliced segment 7/8 Influenza A vmRNAs (Replicate #1) |
| Sample type |
SRA |
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| Source name |
A/Puerto Rico/8/1934(H1N1)
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| Organism |
Influenza A virus |
| Characteristics |
strain: A/Puerto Rico/8/1934(H1N1)
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| Treatment protocol |
Cell pellets were resuspended in 1 ml of RNA probing buffer (RPB) [50 mM HEPES pH 7.9; 140 mM NaCl; 3 mM KCl] and pre-equilibrated at 30 °C for 5 min. 1.76 M DMS in ethanol was added to a final concentration of 50 mM. After gentle vortexing, probing was allowed to proceed at 30 °C for 3 min with moderate shaking. Reactions were quenched by addition of DTT to a final concentration of 0.7 M. Cells were collected at 2000 x g (4 °C) for 3 min and subsequently washed once with PBS/0.7 M DTT.
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| Growth protocol |
Confluent MDCK cells (Sigma Aldrich, #84121903-1VL) in a 10cm cell culture dish were infected with Influenza A virus (A/Puerto Rico/8/1934(H1N1)) at an MOI of 5 in DMEM, 0.14 % BSA and 1 µg/ml TPCK-Trypsin for 6h at 37 °C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected by centrifugation and total RNA extracted with TRIzol Reagent (ThermoFisher Scientific, #15596018). After phase separation, the aqueous phase was added to 1 ml of 100% ethanol and the RNA purified on RNA Clean & Concentrator-5 columns (Zymo Research, #R1016).
RT reactions were performed in 20 µl total reaction volume. An anchored oligo(dT) primer with overhang for subsequent PCR was used at a final concentration of 50 µM. The reaction was allowed to proceed at 57 °C for 2 h. Afterwards, RNA was degraded by adding 1 µl 5 M NaOH and heating to 95 °C for 3 min and subsequently purified on RNA Clean & Concentrator-5 columns. Short splice isoforms were then specifically amplified via PCR and gel purified. The short splice isoforms were mixed equimolarly and fragmented by sonication to yield fragments in the range of 100-300 bp. Sonicated DNA was gel purified to remove residual full-length products and subjected to library preparation using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs, #E6240L) according to manufacturer’s instructions, but omitting size selection.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Influenza A spliced in vivo (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 |
GPL25780 |
| Series (1) |
| GSE122286 |
In vivo analysis of Influenza A mRNA secondary structures identifies critical regulatory motifs |
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| Relations |
| BioSample |
SAMN10392642 |
| SRA |
SRX4993259 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3463239_Spliced_rep1.wig.gz |
12.2 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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