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| Status |
Public on Jan 29, 2024 |
| Title |
HEK293T,mock-PRAISE,rep 1 |
| Sample type |
SRA |
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| Source name |
Epithelial
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Epithelial
cell line: HEK293T
cell type: human embryonic kidney 293 cells
genotype: WT
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| Treatment protocol |
For transfection, 2.5 x 105 of HEK293T cells were seeded on 24-well plates (Corning), and all transfections were conducted when cells reached approximately 80% confluency after 20-24h. The plasmids were extracted using EndoFree Mini Plasmid Kit II (TIANGEN BIOTECH, DP118), and were transfected into cells by Lipofectamine LTX reagent (Invitrogen) supplemented with PLUS reagent (Invitrogen) according to the manufacturer’s instructions.
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| Growth protocol |
HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
total RNA was extracted from cells using TRIzol reagent (Life Technologies)
RNA libraries were prepared using modified eCLIP library construction, NEBNext Small RNA library kits (NEB) and SMARTer Stranded Total RNA-Seq kits (v3) - Pico Input mammalian (Takara) Library Prep Kit following manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MGISEQ-2000RS |
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| Description |
transcriptome-wide quantitative Ψ detection in wildtype HEK293T cells without treatment
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| Data processing |
Analysis was performed as previously reported. Briefly, only read 2 was used for subsequent analysis.
Trim_galore first subjected raw to adaptor trimming and filtering of low-quality reads. Duplicated reads were removed by Seqkit (version 0.13.2).The key parameters were: seqkit rmdup -s. umi_tools (version 1.0.0) was used to remove the 8bp UMI in the deduplication read. Six bases after the UMI added during library construction at the 5’ end of inserted sequences and the six bases at the 3’ end of inserted sequences were also removed, using the key parameters: umi_tools extract --extract-method=string --bc-pattern=NNNNNNNNNNNNNN and umi_tools extract --extract-method=string --3prime --bc-pattern=NNNNNN.
Then the cleaned reads were down-sampled to 40Mb and were mapped to the reference transcriptome(hg38, NCBI Refseq) using PRAISE-tool (https://github.com/Zhe-jiang/PRAISE), and the pseudouridine signals as deletion rate in the transcriptome could be calculated.
We identified putative off-target Ψ sites using the following approach. (1) >15 deletion reads covering each candidate site in restart(+) samples. (2) >100 reads covering each candidate site in restart(-) samples. (3) The deletion rate in restart(-) samples must be less than 1%. (4) The identified off-target Ψ sites must further meet the following criteria: the deletion rate difference in gCtrl, restart v3 and mock groups must be > 20%. (5) For off-target Ψ sites, we applied the statistical test to each of them (contingency table test between restart(+) sample and restart(-) sample), and the P value between the two groups must be <0.000001.
Assembly: GRCh38
Supplementary files format and content: Analysis was performed as previously reported. Briefly, only read 2 was used for subsequent analysis. Trim_galore first subjected raw to adaptor trimming and filtering of low-quality reads. Duplicated reads were removed by Seqkit (version 0.13.2).The key parameters were: seqkit rmdup -s. umi_tools (version 1.0.0) was used to remove the 8bp UMI in the deduplication read. Six bases after the UMI added during library construction at the 5’ end of inserted sequences and the six bases at the 3’ end of inserted sequences were also removed, using the key parameters: umi_tools extract --extract-method=string --bc-pattern=NNNNNNNNNNNNNN and umi_tools extract --extract-method=string --3prime --bc-pattern=NNNNNN.Then the cleaned reads were down-sampled to 40Mb and were mapped to the reference transcriptome(hg38, NCBI Refseq) using PRAISE-tool (https://github.com/Zhe-jiang/PRAISE), and the pseudouridine signals as deletion rate in the transcriptome could be calculated. We identified putative off-target Ψ sites using the following approach. (1) >15 deletion reads covering each candidate site in restart(+) samples. (2) >100 reads covering each candidate site in restart(-) samples. (3) The deletion rate in restart(-) samples must be less than 1%. (4) The identified off-target Ψ sites must further meet the following criteria: the deletion rate difference in gCtrl, restart v3 and mock groups must be > 20%. (5) For off-target Ψ sites, we applied the statistical test to each of them (contingency table test between restart(+) sample and restart(-) sample), and the P value between the two groups must be <0.000001.
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| Submission date |
Jul 18, 2023 |
| Last update date |
Jan 29, 2024 |
| Contact name |
Wenqing Liu |
| E-mail(s) |
puyulwq@163.com, liuwq21@gamils.com
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| Organization name |
Peking University
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| Street address |
No. 5 Summer Palace Road, Haidian District, Beijing
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| City |
Beijing |
| ZIP/Postal code |
010-100871 |
| Country |
China |
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| Platform ID |
GPL30209 |
| Series (2) |
| GSE237632 |
Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons [PRAISE] |
| GSE237633 |
Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons |
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| Relations |
| BioSample |
SAMN36535364 |
| SRA |
SRX21075617 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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