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Status |
Public on Sep 30, 2024 |
Title |
kras_s17n_dna_barcode_AGACATTGCCATAAACGAAG |
Sample type |
SRA |
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Source name |
kidney
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Organism |
Mesocricetus auratus |
Characteristics |
tissue: kidney cell line: BHK-21 cell type: fibroblast cells genotype: repRNA-v4 based treatment: no
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Treatment protocol |
Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. After another 9 d of culture, cells were harvested for total RNA extraction, sequencing, and mutation analysis.
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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.
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Extracted molecule |
total RNA |
Extraction protocol |
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA. The KRAS (S17N)-T2A-PuroR barcode region was amplified from both the DNA library plasmid and the cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Sequel II |
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Data processing |
The resulting Pacbio sequencing data were demultiplexed based on samples and barcodes using in-house Python scripts. The KRAS (S17N) region was aligned to KRAS (S17N) reference sequence using BWA 0.7.17-r1188. Mutation (mismatch) information for each sequencing read was obtained from the bam file using pysam 0.18.0 and custom Python scripts. Assembly: CDS of KRAS (S17N) from H. sapiens Supplementary files format and content: The processed files in txt format contain mutation (mismatch) information for each read.
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Submission date |
Jul 09, 2024 |
Last update date |
Sep 30, 2024 |
Contact name |
Yihan Lin |
E-mail(s) |
yihan.lin@pku.edu.cn
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Organization name |
Peking University
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Street address |
No.5 Yiheyuan Road, Haidian
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City |
Beijing |
ZIP/Postal code |
10087 |
Country |
China |
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Platform ID |
GPL33510 |
Series (2) |
GSE235343 |
Replicative RNA enables directed evolution and Darwinian adaptation in mammalian cells |
GSE271874 |
Tracing the lineage of replicative RNAs in the process of KRAS (S17N) evolution |
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Relations |
BioSample |
SAMN42389391 |
SRA |
SRX25260221 |
Supplementary file |
Size |
Download |
File type/resource |
GSM8387383_kras_s17n_dna_barcode_AGACATTGCCATAAACGAAG.fq.sam.mismatch_positions.txt.gz |
152 b |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
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