|
| Status |
Public on Jan 29, 2024 |
| Title |
MEF, RESTART v3-treated, rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Mus musculus |
| Characteristics |
tissue: embryo
cell line: MEF
cell type: a type of fibroblast prepared from mouse embryo
genotype: W392X
|
| Treatment protocol |
RESTART v3 expressing AAVs were packaged in PackGene Company. 16HBE cells were seeded at a density of 1x105 in 24-well plate (Corning). 24 hours after seeding, 16HBE cells (CFTR-R553X) were transduced by AAV2 at multiplicities of infection 1 x 105. The cell medium was changed 24 hours after transduction. A group of 16HBEge cells was treated with 100 µg/mL G418. 5 days after transduction and 18 hours after G418 treatment, whole cell patch clamp experiments were performed. Besides, MEFs were seeded into 100 mm culture dishes at a density of 1.5 x 105 cells and were transduced by AAVDJ at multiplicities of infection of 5 x 106, 24 hours after seeding. The cell medium was changed 48 hours after transduction, and cells were collected after 7 days.
|
| Growth protocol |
MEF cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C. 16HBEge cells were grown under 5% CO2 at 37°C in MEM medium (Corning) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted from cells using TRIzol reagent (Life Technologies)
RNA libraries were prepared using modified eCLIP library construction, NEBNext Small RNA library kits (NEB) and SMARTer Stranded Total RNA-Seq kits (v3) - Pico Input mammalian (Takara) Library Prep Kit following manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
detect modification level of specific TAG sites in W392X with RESTART v3 treatment
targetseq-CFTRandmIDUA.xlsx
|
| Data processing |
First, sequencing reads with the same unique molecular identifier (UMI) were grouped and UMI groups with less than 3 reads were discarded.
Then, PCR duplicates were removed by retaining the most consensus sequence in each UMI group.
Adapter sequences were further trimmed from deduplicated reads with Trim_galore software (version0.6.7) with default parameters.
The 10-nucleotide UMIs were removed by umi_tools (version 1.0.0).
The cleaned reads were mapped to the targeted sequences using PRAISE-tool and the deletion rate of target sites was calculated.
Assembly: GRCh38
Supplementary files format and content: First, sequencing reads with the same unique molecular identifier (UMI) were grouped and UMI groups with less than 3 reads were discarded. Then, PCR duplicates were removed by retaining the most consensus sequence in each UMI group. Adapter sequences were further trimmed from deduplicated reads with Trim_galore software (version0.6.7) with default parameters. The 10-nucleotide UMIs were removed by umi_tools (version 1.0.0). The cleaned reads were mapped to the targeted sequences using PRAISE-tool and the deletion rate of target sites was calculated.
|
| |
|
| Submission date |
Dec 08, 2023 |
| Last update date |
Jan 29, 2024 |
| Contact name |
Wenqing Liu |
| E-mail(s) |
puyulwq@163.com, liuwq21@gamils.com
|
| Organization name |
Peking University
|
| Street address |
No. 5 Summer Palace Road, Haidian District, Beijing
|
| City |
Beijing |
| ZIP/Postal code |
010-100871 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE237629 |
Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons [Target-seq] |
| GSE237633 |
Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons |
|
| Relations |
| BioSample |
SAMN38735086 |
| SRA |
SRX22835930 |
