|
| Status |
Public on Jul 23, 2023 |
| Title |
Identification of the dedifferentiation trajectory of the cells without Arid1a using single cell RNA-seq |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
The primary mouse hepatocytes were isolated from the liver tissue Arid1af/f mouse. To immortalize mouse primary hepatocytes, the isolated cells were first infected with lentivirus expressing simian virus 40 large-T protein (SV40LT), and then cultured until proliferated cell colonies appear. Cells were further transformed with lentivirus expressing HrasV12D to generate a cell line called AB17, which were cultured in DMEM supplemented with 10% FBS and 100 µg/ml penicillin-streptomycin in a humidified incubator at 37℃ with 5% CO2.To get insight into trajectory of dedifferentiation induced by Arid1a loss, we performed single-cell RNA sequencing (scRNA-seq) on FACS-sorted GFP+ AB17 cells using Fluidigm C1 platform, and obtained the scRNA-seq data from 76 cells with Arid1a knockout induced by Ad-Cre and 73 cells infected with Ad-GFP as control. Significantly, these AB17 cells with or without Arid1a were classified to the two distinct clusters based on their scRNA-seq data via the unsupervised clustering with SC3 software and the dimensionality reduction analysis using t-distributed stochastic neighbor embedding (tSNE) visualization, where the major Ad-Cre infected AB17 cells were assembled into cluster 1 while the majority of AB17 cells with empty Ad-GFP as a control belonged to cluster 2. These data suggest that Arid1a loss obviously dysregulated the gene expression pattern, as represented by the top ten differentially expressed genes between these two clusters.
|
| |
|
| Overall design |
48h after infection with Ad-GFP and Ad-Cre, AB17 cells were sorted to get GFP positive cells. Single cell was captured, reverse-transcribed and amplified on Fluidigm C1 platform. The next generation sequencing was performed by Nextera XT DNA library prep kit (Illumina), followed by sequencing on Illumina X Ten platform using a 150 bp paired-end sequencing strategy. An ERCC Spike-in mix (Ambion) was used as the technical control.
|
| Web link |
https://pubmed.ncbi.nlm.nih.gov/35964817/
|
| |
|
| Contributor(s) |
Han Z, Wang L |
| Citation(s) |
35964817 |
| |
| Submission date |
Nov 25, 2019 |
| Last update date |
Jul 24, 2023 |
| Contact name |
Ze-guang Han |
| E-mail(s) |
hanzg@sjtu.edu.cn
|
| Organization name |
Shanghai Jiao Tong University
|
| Department |
Shanghai Center for Systems Biomedicine
|
| Street address |
800 Dongchuan Road
|
| City |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
| |
|
| Platforms (1) |
|
| Samples (149)
|
|
| This SubSeries is part of SuperSeries: |
|
| Relations |
| BioProject |
PRJNA591558 |
| SRA |
SRP233170 |
Less...
More...