|
| Status |
Public on Jan 14, 2020 |
| Title |
A3_A3SLMut1_293 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293t cells transfected with HIV-1 NHG A3SLMut1
|
| 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 A3SLMut1
|
| 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 |
SAMN11786251 |
| SRA |
SRX5868190 |
