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| Status |
Public on Sep 30, 2024 |
| Title |
EGFP_Post_1st_rep2_571 |
| 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: molnupiravir
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| Treatment protocol |
After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After another 5-7 days, a relatively stable and homogeneous population of polyclonal cells carrying replicative RNA with expression of EGFP or StayGold was generated (i.e., Parent). Approximately 1~2 million cells were passaged into a new 10-cm culture dish and treated with molnupiravir (10 µM) for two consecutive days with daily media changes. Then, they were divided equally into five separate 10-cm dishes and subjected to flow cytometry sorting after an additional cultivation period of approximately 3-4 days (i.e., Post_1st). After about another 7 days, these cells are expected to proliferate to a quantity of around 107. Similarly, these cells were exposed to a two-day treatment with molnupiravir (10 µM) and subsequently transferred onto five new 10-cm dishes for cultivation over a period of 3-4 days. The positive population was then sorted using flow cytometry at a ratio of 0.5%. The sorted cells were cultured to a count of approximately 106 and utilized for cryopreservation and total RNA isolation (i.e., Post_2nd).
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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.
Target genes (e.g., EGFP, StayGold) were amplified from the cDNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and primers specific to each target (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 |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
no
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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 or StayGold
Supplementary files format and content: The processed files in xlsx format contain information such as the number of aligned bases per position, the number of base mutations per mutation type (i.e., A>T, A>G, A>C…), the count of base deletions and insertions, the total number of mapped bases, and the frequency of base mutations.
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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 |
| GSE271876 |
Blue‐shifting green fluorescent proteins using REPLACE |
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| Relations |
| BioSample |
SAMN42389437 |
| SRA |
SRX25260210 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8387403_EGFP_Post_1st_rep2_571_nt_mutation.xlsx |
73.9 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
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