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| Status |
Public on Jun 22, 2022 |
| Title |
MHCC97H_RNC-nanopore |
| Sample type |
SRA |
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| Source name |
MHCC97H cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: liver cancer cells
cell line: MHCC97H
molecule subtype: ribosome nascent-chain complex(RNC)
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| Treatment protocol |
MHCC97H cells were pre-treated with 100 μg/mL cycloheximide (Acmec, Shanghai, China) for 10 min at 37°C, followed by 5 mL pre-colded PBS (Beyotime, Shanghai, China) washes twice and lysis for 30 min on ice by 2 mL pre-cooled human cell lysis buffer (20 mM Tris-HCl, 5 mM MgCl2, 150 mM KCl, 1 mM DTT, 100 μg/mL cycloheximide, 25 units/ml Turbo DNase I, 1% Triton X-100). Cell lysates were clarified by centrifuge at 17000 x g at 4°C for 15 min, supernatants were transferred on the surface of 14.5mL sucrose cushion (30% sucrose, 20 mM Tris-HCl, 5 mM MgCl2, 150 mM KCl, 1 mM DTT, 100 μg/mL cycloheximide). RNCs were purified by ultra-centrifugation in a Type 70Ti rotor (Beckman Coulter, Brea, CA, USA) at 185,000 x g for 5 h at 4°C.
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| Growth protocol |
MHCC97H cells were cultured in the DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 10 μg/mL ciprofloxacin, and both of cells were detected mycoplasma negative during maintenance and upon experiments.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Direct RNA Sequencing Kit (Oxford Nanopore Technologies plc, Oxford, UK) was used for full-length RNC-seq library preparation. The prepared library was load into a MinION flow cell (Oxford Nanopore Technologies plc, Oxford, UK) and sequenced on MinION device (Oxford Nanopore Technologies plc, Oxford, UK).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
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| Data processing |
Base-calling was performed with MinKNOW(V 3.6.5).
Reads were aligned to GRCh38 no-alt analysis set (accession GCA_00001405.15) using minimap2 v2.7-r654 in spliced alignment mode with the command: minimap2 -ax splice -uf -k14 -secondary=no.
FLAIR correct (v1.5) was used to correct the splice-site boundaries of reads.
Genome_build: GRCh38, accession GCA_000001405.15
Supplementary files format and content: Flair correct data contains identified isoforms
Supplementary files format and content: count data
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| Submission date |
Mar 14, 2022 |
| Last update date |
Jun 22, 2022 |
| Contact name |
Gong Zhang |
| Organization name |
Jinan University
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| Lab |
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes
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| Street address |
Jinan University, No.601, West Huangpu Avenue ，Guangzhou, Guangdong, China.
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| City |
guangzhou |
| ZIP/Postal code |
510000 |
| Country |
China |
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| Platform ID |
GPL24106 |
| Series (1) |
| GSE198624 |
Efficient detection of alternative spliced human proteome using translatome sequencing |
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| Relations |
| BioSample |
SAMN26661657 |
| SRA |
SRX14464490 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5953857_MHCC97H_RNC-nanopore.flair.bed.gz |
1.1 Mb |
(ftp)(http) |
BED |
| GSM5953857_MHCC97H_RNC-nanopore_counts.txt.gz |
321.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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