|
| Status |
Public on Jan 01, 2024 |
| Title |
293T_met40 |
| Sample type |
SRA |
| |
|
| Source name |
Cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Cell line
cell line: HEK293T
cell type: Embryonic kidney cells
genotype: WT
treatment: Fixed by 40% methanol
|
| Treatment protocol |
The cultured cells were washed twice with 1ml of ice-cold PBS and resuspended in 200ul of ice-cold PBS, 800ul of methanol were added dropwise and gently mixed.
|
| Growth protocol |
The cells were cultured in RPMI 1640 medium or DMEM supplemented with 10% fatal bovine serum and 1% penicillin–streptomycin. Adherent cells were detached by 0.25% Trypsin-EDTA at 37℃ for 3min and quenched with growth medium. All the cells were maintained in 37℃ with 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The methanol fixed cells were kept in -20℃ for 30min and pelleted at 500×g for 5min at 4℃. After two further washes with 1ml of ice-cold wash-resuspension buffer I, permeabilized cells were resuspended in 20ul of ice-cold wash-resuspension buffer II.
The cells were mixed with 1ul 10mM dNTP and 1ul 100M oligo-dT30VN and incubated at 55C for 5min, then placed on ice immediately for 2min to facilitate RNA denaturation. Next, reverse transcription was performed by adding 6ul RT mix. Full-length transcriptome amplification and quality control. After reverse transcription, cells were further lysed by adding 5ul Proteinase K and incubating at 56C for 60 min and 95C 10 min. Then, the PCR reaction was performed after added 25ul PCR buffer. PCR product was purified by DNA Clean & ConcentratorTM-5 kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
The 40% methanol-fixed cells were mixed with 1ul 10mM dNTP and 1ul 100M oligo-dT30VN and incubated at 55C for 5min, then placed on ice immediately for 2min to facilitate RNA denaturation. Next, reverse transcription was performed by adding 6ul RT mix. Full-length transcriptome amplification and quality control. After reverse transcription, cells were further lysed by adding 5ul Proteinase K and incubating at 56C for 60 min and 95C 10 min. Then, the PCR reaction was performed after added 25ul PCR buffer. PCR product was purified by DNA Clean & ConcentratorTM-5 kit.
|
| Data processing |
The adapter sequences and low-quality reads were removed using fastp.
The processed reads were aligned to the genome using STAR.
The duplicate reads with identical sequences were removed using Picard.
The transcripts were assembled and quantified using HTSeq.
Assembly: hg38;mm10
Supplementary files format and content: Tab-separated matrix files of counts for bulk samples
Supplementary files format and content: HDF5 matrix files of counts for the single-cell samples
|
| |
|
| Submission date |
May 22, 2023 |
| Last update date |
Jan 01, 2024 |
| Contact name |
Yicong Xu |
| E-mail(s) |
maodatou88@hotmail.com
|
| Organization name |
Sun Yat-sen University
|
| Street address |
No. 135, Xingang Xi Road
|
| City |
Guangzhou |
| State/province |
Guangdong |
| ZIP/Postal code |
510275 |
| Country |
China |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE233170 |
The transcriptome profiling of multi-cell types by the droplet based full-length single-cell transcript sequencing |
|
| Relations |
| BioSample |
SAMN35126007 |
| SRA |
SRX20412622 |
