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Sample GSM3782958 Query DataSets for GSM3782958
Status Public on Jan 14, 2020
Title A3_NHG_293-2
Sample type SRA
 
Source name HEK293t cells transfected with HIV-1 NHG
Organisms Homo sapiens; Human immunodeficiency virus 1
Characteristics human cell type or cell line: HEK293t cell line
infection or transfection: transfected with HIV-1 NHG
Treatment protocol DMS-treatment for RNA secondary structure analysis, between 0.5-2% DMS at 37C for 5 minutes. Shake during DMS treatment for suspension cells.
Growth protocol HEK293t were grown in T75 and cultured in DMEM+ 10% FBS + Penn/strep. Transfections were done in 6-well plates with X-tremegene9.
Extracted molecule total RNA
Extraction protocol DMS-treated cells were pelleted and resuspended in 1 mL Trizol, RNA was extracted by phenol-chloroform extraction and isopropanol precipitation.
3' Phosphorylation with rSAP, linker ligation with T4 RNA ligase truncated KQ, excess linker degraded with RecJ and 5'Deadenylase. rRNA was subtracted with RiboZero, and RT was performed with TGIRT-III. cDNAs were isolated by gel extraction on a 10% TBE-Urea gel and extracted by isopropanol precipitation. cDNAs were circularized with circLigase and amplified by PCR with phusion polymerase. PCR products were purified by gel extraction from an 8% TBE gel and isopropanol precipitation. Samples were quantified by Bioanalyzer.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina MiSeq
 
Description DMS-modified RNA
Data processing Library strategy: DMS-MaPseq
Alignment with bowtie2 (2.3.4.1).
Read trimming (TrimGalore v0.4.1) and QC (FastQCv0.11.8).
DREEM Clustering (custom code available at https://gitlab.com/Corbin-Rouskin/RNA_structure/tree/gpu).
RNAstructure (v6.0.1) folding and VARNA (v3.93) visualization.
.sam files generated with bowtie2, .dot files generated by RNAstucture, _Mu.txt files produced by DREEM clustering algorithm.
GRCh38; HIV-1NL4-3 (Genbank accession number AF324493.1); HIV-1NHG(Genbank accession number JQ585717.1)
Supplementary_files_format_and_content: *_Clusters_Mu.txt files produced by DREEM clustering algorithm.
 
Submission date May 20, 2019
Last update date Mar 20, 2020
Contact name Silvi Rouskin
Organization name Whitehead Institute
Street address 455 Main Street
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
 
Platform ID GPL26694
Series (1)
GSE131506 HIV-1 RNA structure and heterogeneity analysis
Relations
BioSample SAMN11786253
SRA SRX5868188

Supplementary file Size Download File type/resource
GSM3782958_A3_NHG_293-2_Clusters_Mu.txt.gz 1.3 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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