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| Status |
Public on May 05, 2019 |
| Title |
DMS-MaPseq on in vivo-purified Influenza A vmRNA (Replicate #2) |
| 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 200 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 |
50 mg of total RNA were used for poly(A)-enrichment with Dynabeads Oligo(dT)25 (Thermo Fisher Scientific, #61002). The isolated poly(A) RNA was used as input for the viral mRNA capture using biotinylated DNA probes (1 probe for each segment with a length of 80 nt complementary to viral mRNA, for sequences refer to Table S1). 100 ml of Dynabeads MyOne Streptavidin T1 beads (ThermoFisher Scientific, #65601) were washed twice with 500 µl Wash Buffer 2 [100 mM NaOH; 50 mM NaCl], twice with 500 µl Wash Buffer 3 [100 mM NaCl], and resuspended in 200 ml 2X Binding & Wash Buffer 1 [10 mM Tris-HCl pH 7.5; 2 M NaCl; 1 mM EDTA]. 5 ml of an equimolar mix of all 8 probes (100 mM pool) in 200 ml water were denatured at 85 °C for 3 min, then immediately placed on ice and added to the washed beads. Probes were immobilized on beads for 20 min at 37 °C, with vigorous shaking. Beads were then washed twice with 500 µl 1x Binding & Wash Buffer 1 and resuspended in 100 µl RNA Binding Buffer [20 mM Tris-HCl pH 7.5; 1 M LiCl; 2 mM EDTA]. To the poly(A)-enriched RNA in 100 µl water, 100 µl RNA binding buffer were added and the sample was denatured at 70 °C for 5 min and immediately placed on ice. Washed beads, supplemented with 40 U of SUPERase In RNase Inhibitor, were then added to the poly(A) RNA and binding was allowed to occur at 37 °C for 2 h, with vigorous shaking. Samples were washed twice with 1 ml of Wash Buffer A (10 mM Tris-HCl pH 7.5; 150 mM LiCl; 1 mM EDTA; 0.1 % SDS] and once with 1 ml Wash Buffer B [10 mM Tris-HCl pH 7.5; 150 mM LiCl; 1 mM EDTA]. Beads were resuspended in 100 µl 95% formamide and 10 mM EDTA pH 8.0, and incubated at 90 °C for 10 min. Eluted RNA was separated from the beads and purified with 1 ml of TRIzol. To remove residual DNA probes, the eluate was treated with DNase I for 15 min at 37 °C and further purified on RNA Clean & Concentrator-5 columns.
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 |
Influenza A in vivo (Replicate #2)
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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 |
SAMN10392645 |
| SRA |
SRX4993256 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3463236_Invivo_rep2.wig.gz |
69.4 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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