|
| Status |
Public on May 09, 2021 |
| Title |
control-STAMP Direct RNA |
| Sample type |
SRA |
| |
|
| Source name |
control-STAMP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
expressing: only APOBEC
sequencing protocol: directly sequenced on a MinION
|
| Treatment protocol |
For STAMP fusion protein expression STAMP stable HEK293T cells were induced with 50ng/ml (low) or 1ug/ml (high) doxycycline in DMEM for 24-72 hours, followed by Trizol extraction and Direct-zol miniprep (Zymo Research) column purification in accordance with manufacturer protocol. Uninduced cells of the same genetic background were used as negative controls. For mTOR perturbation experiments, cells were treated with 100nM Torin-1 or 100nM insulin alongside 1ug/ml doxycycline induction and harvested for RNA after 72 hours 37C incubation.
|
| Growth protocol |
All stable STAMP HEK293T cell lines were generated using human lenti-X HEK293T cells (HEK293XT, Takara Bio) which are derived from transformed female human embryonic kidney tissue. Cells were maintained in DMEM (4.5 g/L D-glucose) supplemented with 10% FBS (Gibco) at 37o C with 5% CO2. Cells were periodically passaged once at 70-90% confluency by dissociating with TrypLE Express Enzyme (Gibco) at a ratio of 1:10.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Trizol extraction (Fisher) and Direct-zol miniprep (Zymo Research) column purification in accordance with manufacturer protocol
Nanopore direct-RNA or direct-cDNA
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
| |
|
| Description |
RNA isolated from HEK293T cells expressing only APOBEC was directly sequenced on a MinION
instrument model: MinION-Mk1B
|
| Data processing |
Raw reads were aligned to hg19 (genomic) or ENSEMBL (cDNA) reference using Minimap2 with the following parameters: "-ax splice -uf -k14". Aligned reads were then passed through Bcftools mpileup command with a "-Q 5" parameter. The pileup files were then filtered for G & C reference positions. For direct-RNA data the strand was inferred based on mapping directionality. For cDNA alignments the strand as assumed to be positive. For direct-cDNA reads aligned to hg19 sites were intersected with an annotation file and strand was assigned based on gene directionality with ambiguous strands being removed. Sites were then further filtered for C positions based on strand. Confidence scores were calculated using manual implementation of SAILOR with a modification rate of 5%. Bed files were generated by filtering SAILOR confidence scores >=0.5. Sites found in the APOBEC-only control were removed from the RBFOX2 files. SNPs were removed by bedtools intersect with dbSNP (v147).
Genome_build: hg19 or ENSEMBL cDNA
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Aug 04, 2020 |
| Last update date |
May 09, 2021 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL24106 |
| Series (2) |
| GSE155708 |
Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [NP] |
| GSE155729 |
Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes |
|
| Relations |
| BioSample |
SAMN15731430 |
| SRA |
SRX8887795 |
