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Sample GSM7500628 Query DataSets for GSM7500628
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.
 
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
 
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

Supplementary file Size Download File type/resource
GSM7500628_T4_mcb152.EGFP_aa_66_203_information.xlsx 13.1 Kb (ftp)(http) XLSX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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