GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4661373

Query DataSets for GSM4661373

Status

Public on Nov 12, 2020

Title

SARS-HKU5-N

Sample type

SRA

Source name

in vitro refolding

Organism

Severe acute respiratory syndrome coronavirus 2

Characteristics

strain: SARS-CoV BtCoV-HKU5

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.

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.

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

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Description

UTR and flanking region

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.

Submission date

Jul 07, 2020

Last update date

Nov 12, 2020

Contact name

Lei Sun

E-mail(s)

sunlei0227@sdu.edu.cn

Phone

13121145408

Organization name

Shandong University