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| Status |
Public on Dec 31, 2022 |
| Title |
ZIKV_CONTROL_rep1 |
| Sample type |
SRA |
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| Source name |
Zika footprinting control
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| Organism |
Zika virus |
| Characteristics |
tissue: viral RNA
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| Treatment protocol |
Eight reactions, four for DENV and four for ZIKV experiment, with 1 µg of vRNA in nuclease-free water were prepared in 0.2-ml PCR tubes. To renature the viral RNA, tubes were heated at 90 °C, for 2 min, in a thermal cycler with heated lid on and then transferred to ice for 2 min. 5 μL of 10× RNA structure buffer were added to the vRNA and gently homogenized by pipetting up and down. Then, tubes were placed into the thermal cycler and temperature increased from 4 °C to 37 °C over 35 min. DENV or ZIKV C proteins were added to the four reactions in increasing concentrations, to achieve different final ratios of C protein monomers to one molecule of vRNA (1:20, 1:100, 1:250 and 1:500). Samples were incubated at 37 °C for 30 min, to allow the interaction of the C protein with vRNA. 5 μl of RNase A/T1 Mix (RNase A 500 U/mL and RNase T1 20000 U/mL; Ambion AM2286) diluted 100 times were added to each tube, and incubated at 37 °C for 30 min. The dilution of RNase A/T1 Mix was previously determined with four serial 10 times dilutions of RNase A/T1 Mix from the stock solution. 45 µL of nuclease-free water were added to each sample to adjust the final volume of the reaction mixtures to 100 µL, and then transferred to 1.5 microfuge tubes with the same volume of phenol–chloroform–isoamyl alcohol. After mixing, tubes were centrifuged at 13,200 × g for 10 min at 4 °C. The aqueous phase of each sample was transferred to a new tube containing 10 µL sodium acetate 3 M and 1 µL glycogen 20 mg/mL. 300 µL of ethanol 100% were mixed with each aqueous solution and, to precipitate the vRNA, samples were stored at -20 °C overnight. To collect the vRNA, samples were spin at 13,200 × g for 30 min at 4 °C. Supernatants were discarded and 800 µL of ethanol 70% were added to the pellet. Samples were spin at 13,200 × g for 15 min at 4 °C and the supernatants removed. A dry spin at 13,200 × g for 1 min at 4 °C was made to completely remove ethanol. vRNA pellets were resuspend in 15 µL of nuclease-free water and stored at -80 °C.
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| Growth protocol |
Following previous procedures,[16] DENV and ZIKV C proteins were expressed in Escherichia coli cells C43 (DE3) and C41 (DE3), transformed with the DENV 2 C protein gene and with the ZIKV Brazil C protein gene, respectively, cloned into a pET21a plasmid (ampicillin resistance). 6 mL LB medium with 100 μg/mL ampicillin were inoculated with a single E. coli colony, containing the pET21a, from LB-agar plates with 100 μg/mL ampicillin, and incubated overnight at 220 rpm, 37 °C. The overnight culture was used to inoculate LB with 100 μg/mL ampicillin, to a final dilution of 1:50. The main culture was incubated at 220 rpm and 37 °C, until the optical density at 600 nm (OD600) reaches 0.8. Protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), at 25 °C overnight and with 220 rpm agitation. The overnight culture was centrifuged at 7000 × g for 40 min at 4 °C. Supernatants were discarded and the cell pellets were resuspended in 1/10 of the volume of the initial culture, in a buffer with 25 mM HEPES, pH 7.4, 0.2 M NaCl, 1 mM EDTA, 5% (v/v) glycerol (Buffer 0.2 M NaCl) and 10 μM protease inhibitor cocktail (Sigma). Cells were lysed by sonication, keeping the suspension on ice, with 5 to 7 pulses (1.5 min) and pauses of 2 min. Sodium chloride was added to the cell lysate to achieve a final concentration of 2 M NaCl, and incubated for at least 1 h, with agitation, at 4 °C. The suspension was centrifuged at 16100 × g for 30 min at 4 °C, and supernatants were collected. Before the first chromatography step, these supernatants were diluted fourfold with Buffer 0.2 M NaCl. The diluted supernatants were applied to a 5 mL HiTrap heparin column (HiTrapTM Heparin HP, GE Healthcare) coupled to an ÄKTA purifier system (ÄKTA explorer, GE Healthcare). The C protein was then eluted performing a NaCl gradient from 0.2 to 2 M NaCl. The fractions corresponding to the C protein were collected and injected in a size exclusion column (S200). C proteins were purified in a buffer with 55 mM KH2PO4 and 550 mM KCl, pH 6.0. DENV C protein purified fractions were concentrated with a Centriprep Centrifugal Filter (Millipore; 3 kDa cutoff) and stored at -80 °C. ZIKV C protein purified fractions were centrifuged at 13,400 × g to remove protein aggregates and the supernatants were stored at -80 °C. The purity of the purified recombinant C protein samples was assessed by 15% SDS-PAGE. Protein concentrations were determined from the sample absorbance at 280 nm, with extinction coefficients calculated from their primary structures (GenBank database: AAC59275.1 and KU497555.1, for DENV and ZIKV, respectively). Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed one peak, with the expected mass of the protein monomer and much lower peaks corresponding to a very low degradation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
2 µl of 10× T4 polynucleotide kinase (PNK) buffer and 2 µL of T4 PNK were added to 14 µL of each vRNA sample. Reaction mixtures were incubated for 4 h at 37 °C. Then, additional 0.5 µL of T4 PNK and 2 µL of 10 mM ATP were added, and the mixture reactions were incubated for 1 h at 37 °C. The final volume of the mixtures was adjusted to 50 µL by adding nuclease-free water. Following, high-quality vRNA samples were prepared with an RNA Clean & Concentrator kit (RNA Clean & Concentrator-5, Zymo Research). Briefly, 100 μL of RNA binding buffer were added to each 50 μL of vRNA sample. Then, an equal volume of ethanol 100% was added and mixed. Samples were transferred to a column in a collection tube and centrifuged for 30 s at 12,000 × g. The flow-through was discarded and 400 μL of the second RNA buffer were added. Viral RNA bound to the columns were washed twice. A dry spin was made to completely remove the washing buffer. Columns were transferred into RNA-free tubes and the vRNA was eluted by adding 8 μL of RNase-free water to the column matrix. After 1 min incubation, columns were centrifuged for 30 s at 12,000 × g. The recovered vRNA was double eluted and samples were storage at -80 °C. Library preparation was made with a NEBNext Multiplex Small RNA Library Prep Set for Illumina kit (New England Biolabs), with small modifications to the protocol. 1 μL of 3’ SR Adaptor was added to 6 μL of the high-quality vRNA samples prepared, in nuclease-free PCR tubes. Reaction mixtures were incubated in a preheated thermal cycler for 2 min at 70 °C, and then transferred to ice. 10 μL of 3’ Ligation Reaction Buffer and 3 μL of 3’ Ligation Enzyme Mix were added to each reaction mixture. Samples were incubated in a thermal cycler for 1 h at 25°C. To prevent the formation of dimers of the remaining 3’ SR Adaptor, 4.5 μL of nuclease-free water and 1 μL of SR reverse transcription (RT) Primer for Illumina were added, and samples were incubated for 5 min at 75 °C, 15 min at 37 °C and 15 min at 25 °C. For the ligation of the 5’ SR Adaptor, an adaptor mix was prepared. For that, 5’ SR Adaptor was incubated in a thermal cycler at 70 °C for 2 min, and then immediately transferred to ice. Following, 1 μL of the denatured 5’ SR Adaptor was mixed with 1 μL of 5’ Ligation Reaction Buffer and 2.5 μL of 5’ Ligation Enzyme Mix. The adaptor mix was added to the samples with 3’ SR Adaptor and incubated in a thermal cycler for 1 h at 25 °C. To perform the RT of the adaptor ligated vRNA samples, 30 μL of these samples were mixed in nuclease-free tube with 8 μL of First Strand Synthesis Reaction Buffer, 1 μL of Murine RNase Inhibitor and 1 μL of ProtoScript II Reverse Transcriptase. Reaction mixtures were incubated at 50 °C for 60 min. Then, RT reactions were inactivated at 70 °C for 15 min and stored at -20 °C. A large-scale PCR amplification was performed using 12 PCR cycles, the number of cycles identified as enough to construct a library. For that, 10 μL of the cDNA were mixed, on ice, with 2.5 μL of F-primer (SR primer), 2.5 μL of R-primer (with different barcodes for each sample), 5 μL of nuclease-free water and 20 μL of 2X Q5 Master Mix. The barcoded cDNA samples were normalized to 1000 ng/mL and sent to next generation sequencing on the Illumina platform.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Zika footprinting control replicate 1
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| Data processing |
illumina adapter removal using cutadapt
alignment with bowtie2 'very-sensitive-local' preset
calculation of SPMR from raw read counts
Genome_build: EU081177; KU497555
Supplementary_files_format_and_content: csv: nucleotide_coodinate, singal_per_million_reads (SPMR)
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| Submission date |
Apr 28, 2021 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Roland G Huber |
| E-mail(s) |
rghuber@bii.a-star.edu.sg
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| Phone |
+6564788321
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| Organization name |
Agency for Science, Technology and Research (A*STAR)
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| Department |
Bioinformatics Institute (BII)
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| Lab |
Structure and Function of RNA
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| Street address |
30 Biopolis Street
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| City |
Singapore |
| ZIP/Postal code |
138671 |
| Country |
Singapore |
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| Platform ID |
GPL24221 |
| Series (1) |
| GSE173463 |
Flavivirus Capsid Proteins Facilitate Genome Compaction and Packaging |
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| Relations |
| BioSample |
SAMN18901227 |
| SRA |
SRX10694696 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5268199_RHH5774_SPMR.csv.gz |
73.5 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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