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Status |
Public on Nov 12, 2020 |
Title |
SARS-HKU5-D |
Sample type |
SRA |
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Source name |
in vitro refolding
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Organism |
Severe acute respiratory syndrome coronavirus 2 |
Characteristics |
strain: SARS-CoV BtCoV-HKU5
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Treatment protocol |
For in vivo probing of RNA, NAI-N3 was added to the cell pellet at a final concentration of 100mM and then incubated at 37 ℃ for 5 min with mixing gently. For preparing negative control samples, an equal amount of DMSO (25μl, Sigma-Aldrich, cat. D2650) was added to the cell pellet.
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Growth protocol |
Huh7.5.1 cells were provided by Dr. Yang (Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College), and were maintained at 37°C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, cat.C11965500BT) supplemented with 10% fetal bovine serum (FBS, HyClone, cat.SH30396.03) and penicillin-streptomycin (GENOM, cat.GNM15140). SARS-CoV-2, Isolate IPBCAMS-YL01/2020 was obtained from a clinical sample at Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, which was passaged thrice in Vero cells (ATCC, CCL-81) before use. Infectious titers of SARS-CoV-2 were determined by plaque assay in Vero cells. For SARS-CoV-2 infection, Huh7.5.1 cells were cultured in T-175 flasks at a density of 5×106 cells for 16 h. The cells were briefly washed with DMEM and incubated with SARS-CoV-2/IPBCAMS-YL01/2020 for 1 h at a multiplicity of infection (MOI) of 0.05, then supplemented with DMEM maintenance medium, which was supplemented with 1% BSA(Sigma-Aldrich, cat.B2064)and penicillin-streptomycin, and then cultured at 37°C, 5% CO2 for additional 30 h. Cultured cells were washed twice with PBS before collecting by cell scraper. All experiments involving live SARS-CoV-2 in these studies were performed in a biosafety level 3 facility.
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Extracted molecule |
total RNA |
Extraction protocol |
After probing, samples were transferred immediately to ice in order to stop the reaction. After which it was centrifuged for 5 min at 500 ×g (4 ℃) and discarded the supernatant, cell pellets were resuspended in 6 mL TRIzol (Invitrogen, cat.15596018) and supplemented with 1/5 volumes of chloroform. The sample was vigorously vortexed for 15 sec and then incubated for 5 min at room temperature, after centrifuged for 15 min at 12,000 ×g (4 ℃), the upper aqueous phase was transferred to a clean 15 mL tube, then supplemented with 2 volumes of 100% ethanol and mixed up and down, followed by column purification according to the manufacturer’s instructions (Hipure RNA pure Micro Kit, Magen, cat.R2144-03). We isolated poly(A) RNA with the DynabeadsTM mRNA DIRECT TM kit as the manufacturer’s instructions with the next modification. Wash the ploy-dT beads with washing buffer B for 2 times after the first round of ploy(A) purification. Then perform a second ploy(A) enrichment using the beads for the first-round enriched ploy(A) RNA. Usually, about 1% ploy(A) RNA of the DMSO treated samples and 5‰ ploy(A) RNA of the NAI-N3 treated samples were yield.icSHAPE library were constructed from in vivo modified, in vitro modified or DMSO treated control RNA as previously described with the following modification. We designed the new library linker, reverse transcription (RT) primer, P5 and P7 amplification primer as previous description to adapt to the Illumina hiseq X system. To simplify the library construction of UTR regions of many virus types, we merged the in vitro transcription RNA of different virus into one group according to their sequence variability. i.e. RNA of SARS-CoV-2 (SARS2-C mutation), human NL63 (hNL63) and human HKU1 (hHKU1) was merged into a group (SARS2-C-NL63-HKU1). RNA of SARS and bat HKU5 (bHKU5) was merged into a group (SARS-HKU5). RNA of MERS and bat HKU9 was merged into a group (MERS-HKU9). RNA of SARS-CoV-2 with T mutation (SARS2-T) was a group independently. Libraries of virus infection (modification in vivo, refolding and modification in vitro) was sequenced on Hiseq X system to approximately 200 million reads per replicates while libraries of virus for UTR regions were sequenced about 10-30 million reads per replicates. Though the reads were pair-end sequencing, only the R1 reads (*_1.fastq) which include the reverse transcription stop (RT stop) site were used to for further analysis in this study. icSHAPE
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
HiSeq X Ten |
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Description |
UTR and flanking region 293_np_vitro.out
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Data processing |
remove duplicete reads as icshape pipeline trimmed reads adaptor as icshape pipeline mapping reads to SARS-CoV2 genome or other virus UTR regions or human hg38 with STAR as default parameters calculate rpkm as icshape pipeline calulate reverse transcript stop sites as icshape pipeline calculate icSHAPE score as icshape pipeline Genome_build: SARS-CoV2, SARS2,SARS,MERS,HKU1,HKU5,HKU9,NL63 UTR regions, human hg38 Supplementary_files_format_and_content: tab-delimited text files for icshape files. First column is the Ensemble ID, second is gene length, third is RPKM, then every column is the nucleotide resolution icSHAPE score. NULL means no confident score.
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Submission date |
Jul 07, 2020 |
Last update date |
Nov 12, 2020 |
Contact name |
Lei Sun |
E-mail(s) |
sunlei0227@sdu.edu.cn
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Phone |
13121145408
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Organization name |
Shandong University
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Lab |
Sun Lab
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Street address |
Shandong street
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City |
Qingdao |
State/province |
Shandong |
ZIP/Postal code |
266071 |
Country |
China |
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Platform ID |
GPL28840 |
Series (1) |
GSE153984 |
RNA secondary structome of SARS-CoV-2 and other 6 virus structures in the UTR regions |
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Relations |
BioSample |
SAMN15469620 |
SRA |
SRX8684084 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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