|
| Status |
Public on Jul 07, 2024 |
| Title |
KRAS_invitroRNA |
| Sample type |
SRA |
| |
|
| Source name |
kidney
|
| Organism |
Mesocricetus auratus |
| Characteristics |
tissue: kidney
cell line: BHK-21
cell type: fibroblast cells
genotype: expressing replicative RNA repRNA-v4-KRAS (S17N)
treatment: No
|
| Treatment protocol |
Medium was replaced with fresh medium containing 5 μg/ml puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), 2 μM of molnupiravir was added to the medium. The cells were continued to be cultured and selected in medium with molnupiravir and puromycin for 10 days.
|
| Growth protocol |
BHK-21 cells were cultivated in Minimum Essential Medium with Earle's Balanced Salts (MEM/EBSS) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/ml streptomycin, and 1% MEM Non-Essential Amino Acids Solution (Gibco, #11140050) in a humidified 37℃ incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Each sample collected ~ 1 million cells for RNA isolation using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). 3μg genomic RNA or 500 ng in vitro transcribed RNA was used for reverse transcription.
The KRAS (S17N) DNA or KRAS (S17N) cDNA mutant libraries were subjected to 28 cycles of PCR amplification using KRAS gene specific primers containing Illumina sequencing indexes (resulting in 330 bp amplicons). The PCR products were purified and used for sequencing on the Illumina platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
No
|
| Data processing |
The paired-end sequencing data were initially processed using Trimmomatic 0.39 to remove the 25-bp primer sequences used for PCR amplification and to discard sequences with an average sequencing quality score below the Q36 threshold. The filtered sequences were then aligned to the coding region of KRAS (S17N) using BWA 0.7.17-r1188 software. In order to analyze mismatch variations across different samples, reads containing base deletions or insertions were excluded. Subsequently, an in-house Python script was employed to tally the types of variants and the frequency of mutations per sequencing reads from the resulting BAM files.
Assembly: human KRAS (S17N) coding sequence
Supplementary files format and content: *mutants.number.remove.dis.xlsx: number of mutants for each samples
Supplementary files format and content: KRAS_mutation_number_250bp_all_samples.xlsx: frequency of mutation numbers of sequencing reads for each samples
|
| |
|
| Submission date |
Jun 20, 2023 |
| Last update date |
Jul 07, 2024 |
| Contact name |
Yihan Lin |
| E-mail(s) |
yihan.lin@pku.edu.cn
|
| Organization name |
Peking University
|
| Street address |
No.5 Yiheyuan Road, Haidian
|
| City |
Beijing |
| ZIP/Postal code |
10087 |
| Country |
China |
| |
|
| Platform ID |
GPL28997 |
| Series (2) |
| GSE235327 |
Analyzing the process of mutation accumulation in KRAS evolution experiment using amplicon sequencing |
| GSE235343 |
Replicative RNA enables directed evolution and Darwinian adaptation in mammalian cells |
|
| Relations |
| BioSample |
SAMN35813890 |
| SRA |
SRX20733036 |
