|
Status |
Public on Jul 07, 2024 |
Title |
T4_mcb152 |
Sample type |
SRA |
|
|
Source name |
kidney
|
Organism |
Mesocricetus auratus |
Characteristics |
tissue: kidney cell line: BHK-21 cell type: fibroblast cells genotype: harboring barcoded repRNA-v4 library treatment: No
|
Treatment protocol |
Twenty-four hours post-electroporation (referred to as T1), the medium was replaced with fresh medium containing 5 μg/ml puromycin. After four days of selection (referred to as T5), 10 μM of molnupiravir was added to the medium for a 2-day treatment. Subsequently, the cells were allowed to grow for four days (referred to as T3) and then subjected to flow cytometry sorting. The sorted cells were cultured for approximately one week (referred to as T4) and subsequently treated with molnupiravir for another 2 days (referred to as T5).
|
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 was used for reverse transcription. The extracted RNA was subjected to reverse transcription, followed by amplification using primers specific to genomic RNA targeting the EGFP-PuroR-barcode region. The purified PCR products underwent adapter ligation, fragment selection, primer and polymerase binding, and were then sequenced using a PacBio Sequel IIe instrument to obtain Circular Consensus Sequencing (CCS) reads.
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|
|
Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Sequel II |
|
|
Description |
no
|
Data processing |
The resulting sequencing data were demultiplexed based on samples and barcodes, and the EGFP region was extracted for alignment using pbmm2 1.10.0. Mutation (mismatch) information for each sequencing read was obtained from the bam file. A custom Python script was used to quantify the mutation information at Y66 and T203 within the EGFP region. Assembly: EGFP_PuroR.fa Supplementary files format and content: *EGFP_aa_66_203_information.xlsx: mutation information for aa 66 and 203 of EGFP of each barcodes for each samples Supplementary files format and content: GFP_region_mismatch_number_using_barcode_data_sorted.xlsx: frequency of mutations per sequencing reads for each samples
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|
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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 |
GPL33510 |
Series (2) |
GSE235338 |
Analysis of the accumulation of mutations in self-replicating RNAs using barcoded EGFP library |
GSE235343 |
Replicative RNA enables directed evolution and Darwinian adaptation in mammalian cells |
|
Relations |
BioSample |
SAMN35807158 |
SRA |
SRX20728718 |