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Status |
Public on Sep 30, 2024 |
Title |
repRNA_v4_post_day7_rep2 |
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 |
After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages.
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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 were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA or IVT 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. EGFP region was amplified from the cDNA or DNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and specific primers(For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing using Tsingke Biotechnology's Fast NGS service. In brief, the experimental workflow provided by Tsingke Biotechnology involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina MiSeq |
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Data processing |
Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position. Assembly: Coding sequence of EGFP Supplementary files format and content: The processed files in csv format contain the total mutation frequency for each sample.
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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 |
GPL29575 |
Series (2) |
GSE235343 |
Replicative RNA enables directed evolution and Darwinian adaptation in mammalian cells |
GSE271880 |
Quantification of basal RNA mutation rate in the repRNA-v4 EGFP region |
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Relations |
BioSample |
SAMN42389399 |
SRA |
SRX25260199 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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