|
| Status |
Public on Apr 22, 2020 |
| Title |
TOD_synthetic |
| Sample type |
SRA |
| |
|
| Source name |
chemically synthesized
|
| Organism |
synthetic construct |
| Characteristics |
sample type: in vitro transcribed
library type: pooled
adapter strategy: direct ligation
rna modification: Methylation
|
| Treatment protocol |
Methylations were introduced during the chemical synthesis with the addition of methylated monomers at the appropriate synthesis cycle.
|
| Growth protocol |
RNA was chemically synthesized using 2′-tBDSilyl protected phosphoramidite monomers.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Chemically synthesized RNA was desalted with a Gel-Pak 1.0 desalting column (Glen Research) and concentrated with a SpeedVac (Thermo Fisher Scientific) on low heat. Concentrated RNA was checked for appropriate molecular weight with the Voyager-DE STR Biospectrometry Workstation (Applied Biosystems) and a MALDI-TOF mass spectrometer (Applied Biosystems). Quality-verified RNA was finally size selected using a denaturing 10% PAGE gel.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Raw sequencing files were demultiplexed using novobarcode.
For M2-seq analysis of the HIV 3' UTR construct, the demultiplexed files were run through the ShapeMapper software, and the resulting mutational profile strings were run through the M2-seq analysis pipeline (further details can be found at: https://github.com/ribokit/m2seq) to yield the final two-dimensional reactivity files.
For polyA length analysis, raw demultiplexed reads were analyzed with custom python scripts that directly searched for polyAs surrounded by constant flanking regions. For more information see: https://github.com/DasLab/Anomalous_polyA_RT.
For reactivity analysis, the demultiplexed reads were analyzed with the RNAFramework, ShapeMapper, and ShapeMapper2 pipelines. Custon python scripts were then used to compare computed reactivities between packages.
For more information see: https://github.com/DasLab/Anomalous_polyA_RT.
genome build: GGGTACAAAAAAAAAAAGTACAAAGAAACAACAACAACAAC
Supplementary_files_format_and_content:
For HIV 3'UTR M2-seq analysis: mutation strings indicating the location of mutations for each read are provided in the format of one read per line in a one-hot encoding. Two-dimensional reactivity files are provided in the RDAT file format as specified here: https://rmdb.stanford.edu/deposit/specs/.
For polyA length analysis, CSV tables are provided for each construct, with each column representing data relevant to a single condition (e.g. reverse transcribed with TGIRT). Values in each cell represent the raw number of reads corresponding to a given polyA length. For insertion/substitution tables, the frequency of an insertion or substitution at each nucleotide along the polyA tail (read in 3' to 5' direction).
For reactivity analysis, each plain text file contains the reactivity of a construct for a given condition. Each line contains a reactivity score of a single nucleotide position.
|
| |
|
| Submission date |
Apr 21, 2020 |
| Last update date |
Apr 23, 2020 |
| Contact name |
Rhiju Das |
| E-mail(s) |
rhiju@stanford.edu
|
| Organization name |
Stanford University School of Medicin
|
| Department |
Biochemistry
|
| Lab |
Das
|
| Street address |
279 Campus Dr, B419 Beckman Center
|
| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL17769 |
| Series (1) |
| GSE149061 |
Anomalous reverse transcription through chemical modifications in polyadenosine stretches |
|
| Relations |
| BioSample |
SAMN14655870 |
| SRA |
SRX8151469 |
