|
| Status |
Public on Jan 14, 2020 |
| Title |
9666_RRECD4 |
| Sample type |
SRA |
| |
|
| Source name |
HIV-1 NL4-3 infected CD4 + T cells
|
| Organisms |
Homo sapiens; Human immunodeficiency virus 1 |
| Characteristics |
human cell type or cell line: CD4+ T Cells
molecule subtype: DMS-modified RNA
|
| Treatment protocol |
DMS-treatment for RNA secondary structure analysis, between 0.2-2% DMS at 37C for 5 minutes
|
| Growth protocol |
CD4+ T cells were grown in T75 flasks and cultured in RPMI1640 + 10%FBS + Penn/strep, maintained at a concentration of ~1-3million/mL. CD4+ T cells were activated with 10ug/mL PHA and 100U/mL IL-2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DMS-treated cells were pelleted and resuspended in 1 mL Trizol, RNA was extracted by 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 gelextraction 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 |
| |
|
| 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
Genome_build: GRCh38; HIV-1NL4-3 (Genbank accession number AF324493.1)
Supplementary_files_format_and_content: .sam files generated with bowtie2, .dot files generated by RNAstucture, _Mu.txt files produced by DREEM clustering algorithm. Multiple Mu files were generated for RREmutA and RREmutB by computationally mixing reads from the two samples in specified proportions in order to validate the ability of DREEM to accurately deconvolute the separate structures.
|
| |
|
| Submission date |
Jun 18, 2019 |
| Last update date |
Jan 14, 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 |
SAMN12085966 |
| SRA |
SRX6087067 |
