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| Status |
Public on Jul 25, 2024 |
| Title |
HeLa cells, sh-MePCE-3 |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: cell line
cell line: HeLa
cell type: epithelial cell
genotype: MePCE knockdown
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| Growth protocol |
HeLa cells were maintained in DMEM (Gibco) supplemented with 10% heat-inactivated foetal bovine serum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the pellets (1×107 cells) using Ambion TRIzol® Reagent according to the manufacturer’s instructions, followed by DNase I treatment at 37°C for 60 min.
Ligation reactions contained 2.0 µg total RNA, 1×T4 RNA ligase buffer, 5% PEG8000, 1 U T4 RNA ligase I, and 5 μM 3′ linker (CKOligo-666).
The ligation reaction mixture was incubated at 16°C for 18 hours and inactivated at 65°C for 15 minutes.
20 μl of reverse transcription reaction contained 1X ligation reactions, 1× SSIV buffer, 0.1 μM RT primer (CKOligo-667), 0.5 μM dNTPs, 5 mM DTT, RNase inhibitor, and SuperScript™ IV Reverse Transcriptase was incubated at 50°C for 10 minutes.
A total of 10 U E. coli RNase H was directly added into the reverse transcription reaction, followed by incubation at 37°C for 20 minutes and then heat-inactivation at 70°C for 15 minutes.
The first round PCR was performed with 0.5 μM CKOligo-667 and CKOligo-668 in 50 μl reactions containing 1× Phusion HF buffer, 0.2 mM dNTPs, and 0.02 U Phusion Hot Start II High Fidelity DNA polymerase. First-round PCR products were purified by PCR purification kit.
For the second-round PCR reactions, 5 μl of first-round PCR product was used as a template. 0.5 μM CKOligo-669 as the forward primer for all samples and reverse primers CKOligo-676 (sh-Luc), CKOligo-685 (sh-PARN), CKOligo-686 (sh-LARP7), and CKOligo-687 (sh-MePCE) were used. Amplicons were purified by PCR purification kit (TOOLS).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq X Plus |
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| Data processing |
The library samples were sequenced on the Novaseq X plus using RapidSeq-150 bp pair-end reads.
A 10-nt molecular barcode was used to remove PCR duplicates. After quality filtering, 4.7 to 6.1 million reads were analyzed per sample.
To pass the filter, a read required a minimum match of 20 nts to hTR reference sequence and 10 nts to the linker sequence.
For each filtered read, the most 3′ hTR reference coordinate between hTR:366–641 was identified by matching 20 nts closest to the 3′ end allowing for two mismatches not including the two most 3′ bases.
Assembly: hg38
Supplementary files format and content: excel file includes the raw counts
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| Submission date |
Mar 27, 2024 |
| Last update date |
Jul 25, 2024 |
| Contact name |
Chi-Kang Tseng |
| E-mail(s) |
[email protected]
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| Phone |
+886914101877
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| Organization name |
National Taiwan university, College of Medicine
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| Department |
Graduate institute of microbiology
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| Lab |
742
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| Street address |
No.1 Jen Ai road section 1
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| City |
Taipei |
| ZIP/Postal code |
10051 |
| Country |
Taiwan |
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| Platform ID |
GPL34284 |
| Series (1) |
| GSE262590 |
LARP3, LARP7, and MePCE are Involved in the Early Stage of Human Telomerase RNA Biogenesis |
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| Relations |
| BioSample |
SAMN40628827 |
| SRA |
SRX24074697 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8172113_HeLa-MePCE-3.tsv.gz |
65.2 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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