title: GSM4661371: MERS-HKU9-N; Middle East respiratory syndrome-related coronavirus; OTHER
library_construction_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
attributes.tag: GEO Accession
attributes.value: GSM4661371