|
| Status |
Public on Feb 27, 2019 |
| Title |
scRNAseq in EB+48hrDox(iAscl1-v5 or iNeurog2 Tubb3::GFP) |
| Sample type |
SRA |
| |
|
| Source name |
ES-derived embryoid bodies with induced transcription factors
|
| Organism |
Mus musculus |
| Characteristics |
genotype: pooled Ainv15(iAscl1.V5) and Ainv15(iNeurog2 Tubb3::GFP)
treatment: Dox induction + 48 hours
cell type: Embryoid Bodies
|
| Treatment protocol |
After two days, the EBs were passaged 1:2 and expression of the transgenes was induced by 3 ug/ml Doxycycline (Sigma D9891) to the AK medium. For RNA-seq experiments 2-3x10^5 cells were plated in each 100 mm untreated dishes (Corning).
|
| Growth protocol |
The inducible mESCs were grown in 2i (2-inhibitors) based medium (Advanced DMEM/F12: Neurobasal (1:1) Medium (GIBCO), supplemented with 2.5% mESC-grade fetal bovine serum (vol/vol, Corning), N2 (GIBCO), B27 (GIBCO), 2mM L-glutamine (GIBCO), 0.1 mM ß-mercaptoethanol (GIBCO), 1000 U/ml leukemia inhibitory factor (Millipore), 3mM CHIR (BioVision) and 1 mM PD0325901 (Sigma) on 0.1% gelatin (Milipore) coated plates at 37°C, 8% CO2. To obtain embryoid bodies (EBs), 60-70% confluent mESCs were dissociated by TrpLE (Gibco) and plated in AK medium (Advanced DMEM/F12: Neurobasal (1:1) Medium, 10% Knockout SR (vol/vol) (GIBCO), Pen/Strep (GIBCO), 2mM L-glutamine and 0.1mM ß-mercaptoethanol) on untreated plates for two days (day -2) at at 37°C, 8% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single-cell RNA-seq: Cells (iAscl1-v5 and iNeurog2 Tubb3::GFP) were collected 48 hours after Dox induction and washes were done in 1X PBS with 0.04 mg/ml BSA (Thermo Fisher Sci AM2616). Cells were strained with CellTrics 30 µM (Cat #04-004-2326) to remove cell clumps. Equal number of iA and iN Tubb3::GFP cells were pooled to have 1000 cells/ul.
10X Genomics Chromium Single Cell 3’ library kit was used to generate single cell library for a targeted cell recovery rate of 10.000 cells (120262 Chromium i7 Multiplex Kit, 120236 Chromium Single Cell 3' Chip Kit v2, 120237 Chromium Single Cell 3' Library & Gel Bead Kit v2). Fragment length distribution of the library was determined by Agilent High Sensitivity DNA D1000 Screentape (5067- 5585) system and the final quantification of the library before pooling was done with KAPA library amplification kit (Roche Lightcycler 480). The libraries were sequenced on Illumina NextSeq 500 High Output using V2.5 chemistry with 26x98 bp - 150 cycles run confirmation at the genomics core facility at NYU.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
A list of genotype-specific barcodes was not produced. Submitter pooled the genotypes and identified genotypes based on gene expression markers.
|
| Data processing |
R1 is cell barcode (16bp) followed by UMI (10bp) R2 is the RNA read (98bp) I1 is the sample index (8bp)
Fastq files were generated by using CellRanger (version 2.1.0) mkfastq from 10X Genomics with default settings (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger).
Feature counts: We added the transgene sequences to the reference genome manually to distinguish the two pooled cell lines: V5 (iAscl1) and GFP (iNeurog2 Tubb3::GFP) exogenous sequences were added to the end of chromosome 1 in FastA and GTF files of the mouse reference genome (mm10). A custom reference genome was generated by CellRanger mkref function by passing the modified FastA and GTF files. CellRanger count function was used to generate single cell feature counts for the library.
Downstream analysis: Downstream analysis and graph visualizations were performed in Seurat R package76 (version 2.3.4). Briefly, we removed the cells that have unique gene counts greater than 6800 (potential doublets) and less than 200. After removing the unwanted cells, we normalized the data by a global-scaling normalization method (LogNormalize) with the default scale factor (10000). Linear dimensional reduction was performed by PCA and the clustering was performed by using the statistically significant principal components (identified by jackStraw method and by standard deviation of principle components). The results were visualized by tSNE plots.
Genome_build: mm10
Supplementary_files_format_and_content: HDF5 format raw gene expression matrix from 10X Genomics cellranger count v2.1.0
|
| |
|
| Submission date |
Jan 24, 2019 |
| Last update date |
Feb 28, 2019 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
|
| Phone |
814-865-3008
|
| Organization name |
Penn State University
|
| Department |
Biochemistry & Molecular Biology
|
| Lab |
Shaun Mahony
|
| Street address |
404 South Frear Bldg
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE114176 |
Molecular basis of neuronal subtype bias introduced by proneural factors Ascl1 and Neurog2 |
| GSE125620 |
Molecular basis of neuronal subtype bias introduced by proneural factors Ascl1 and Neurog2 (single-cell RNA-seq) |
|
| Relations |
| BioSample |
SAMN10815725 |
| SRA |
SRX5290059 |
