|
| Status |
Public on Apr 30, 2023 |
| Title |
Single cell RNAseq of FACS sorted VitA- Col1a1+ cells from DEN+WAD mouse liver. |
| Sample type |
SRA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Liver
cell line: Disease state
cell type: Hepatic stellate cell
genotype: Rosa26mTmG;Col1a1-Cre
treatment: diethyl nitrosamine (i.p)+ Western alcohol diet (fed)
|
| Treatment protocol |
Male Rosa26mTmG; Col1a1-Cre (mTmG;CC) mice on C57BL/6J background were injected with diethyl nitrosamine (DEN, 10mg/kg) or saline at 2-wk old and fed for 5 months from 6-wk old liquid Western alcohol diet (WAD) containing ethanol (3.5%v/v) or chow.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Liver non-parenhcymal cells were isoalted by in-situ liver perfusion with pronase and collagenase and vitamin A+ (VitA+) and VitA- Col1a1-expressing cells were collected via FACS on ARIA IIu (Becton Dickinson) with solid state laser excited at 488nm and measured with 510/20BP emission mirrors for GFP and excited at 355nm and measured with 450/50BP mirrors for VitA and data were analyzed by FlowJo 10.8.1 version. The cells were immediately bar-coded for scRNA-seq.
The library was prepared immediately after cell sorting with the target cell number of 10,000 and validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 Fluorometer (Invitrogen) and qPCR (KAPA Biosystems) according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and sample index were added.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
samples were processed through 10x Genomics Single Cell 3' v3.1 following manufacturer protocol. Sequencing data was package using 10x Genomics CellRanger.
|
| Data processing |
The samples were sequenced using a 2x150 Paired End configuration on the Illumina HiSeq
Raw sequencing data were converted to fastq files and de-multiplexed using the 10x Genomics’ Cell Ranger software version 3.1.0.
UMI and cell barcode de-convolution and mapping to the mm10 reference genome were performed with the software to generate the final digital gene expression matrices and cloupe files using the Cell Ranger count command with default parameters.
The cells which passed the quality assessment by a barcode rank graph, were subjected to sequencing and the data analysis was performed with 10X Genomics’ Loupe Browser software.
Assembly: mm10
Supplementary files format and content: Cell Ranger h5 matrix file
|
| |
|
| Submission date |
Apr 28, 2023 |
| Last update date |
May 19, 2023 |
| Contact name |
Hidekazu Tsukamoto |
| E-mail(s) |
htsukamo@med.usc.edu
|
| Organization name |
University of Southern California
|
| Department |
Pathology
|
| Lab |
Southern California Research Center for ALPD & Cirrhosis
|
| Street address |
1333 San Pablo Street MMR414
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90089 |
| Country |
USA |
| |
|
| Platform ID |
GPL21273 |
| Series (1) |
| GSE230843 |
Hepatic stellate cell stearoyl co-A desaturase activates leukotrien B4 receptor 2-β-catanin cascade to promote liver tumorigenesis. |
|
| Relations |
| BioSample |
SAMN34416480 |
| SRA |
SRX20430922 |
