|
| Status |
Public on Oct 23, 2023 |
| Title |
Vagal-derived cells, post-umbilical gut replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
E10 post-umbilical gut and associated ganglion
|
| Organism |
Gallus gallus |
| Characteristics |
tissue: E10 post-umbilical gut and associated ganglion
cell type: vagal chick neural crest
genotype: Wildtype
treatment: Replication incompetent avian retrovirus carrying nuclear (H2B) YFP, injected into the neural tube at somite level 1-7 at embryonic day 1.5. FAC sorted for YFP+ cells
|
| Treatment protocol |
Viral solution was supplemented with 0.3ml of 2% food dye (Spectral Colors, Food Blue 002, C.A.S# 3844-45-9) as an indicator, injected to fill the neural tube between somite 1-7 at HH10 to label vagal neural crest and/or posterior to somite 28 at HH17 to label sacral neural crest in ovo. Embryos were supplied with Ringer’s Solution (0.9% NaCl, 0.042%KCl, 0.016%CaCl2 · 2H2O wt/vol, pH7.0), sealed with surgical tape, and incubated at 37 °C until embryonic day 10.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
At embryonic day 10, the gastrointestinal tract, including the associated Nerve of Remak and pelvic plexuses , was dissected from chick embryos and washed with Ringer’s solution. Pre- and post-umbilical regions were separated, broke into pieces in chilled DPBS and loose-fit homogenized in Accumax solution (EMD Millipore). 400ml of Accumax-tissue mixture was aliquoted into 1.7 ml Eppendorf tubes and shaken at 37 °C for 12mins. After dissociation, chilled Hanks Buffered Saline Solution (HBSS) supplemented by BSA (125mg in 50ml, Sigma; 0.2% w/v) and 1M HEPES (500ml in 50ml, PH7.5, ThermoFisher) was added to quench the reaction. The dissociated cells were passed through a 70mm cell strainer (Corning) and collected by centrifuging at 500g for 11mins at 4 °C. The cells were resuspended in HBSS-BSA, supplemented 7-AAD Viability Staining Solution (Biolegend # 420404, 500 TESTS), and sorted for YFP+, viable single cells using Sony SY3200 cell sorter at the Caltech Flow Cytometry Facility.
Library contrusted per manufacturers instructions. The library was sequenced on NovaSeq S4 lane with 2x150bp reads by Fulgent Therapeutic
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10X Genomics; cells with >2 counts of viral promoter transcript were used for downstream analysis (single_cell_cellranger_RIA_pos.rds)
WEIYI-2-1
|
| Data processing |
Fastq raw data as aligned to standard ENSEMBL galgal6 reference database. Single-cell level gene quantification was then performed using Cellranger v3.1.023 and kallisto 0.46.2 & bustools 0.40.0 pipelines24 with default parameters. Gene count matrices from all the samples were combined and only cells with more than 200 genes detected were kept for the downstream analysis.
DoubletFinder 2.0.3 package was used to remove doublets.
Cells with greater than 2 transcripts of the RIA retrovirus promotor mRNA were then selected for further analysis
The first 30 principal components from PCA analysis were used to find neighbors with Findneighbors function before cell clustering with FindClusters function (resolution = 0.3). UMAP dimensionality reduction was performed using RunUMAP function with uwot-learn selected for the parameter umap.method.
Assembly: galgal6
Supplementary files format and content: R Data Serialization
|
| |
|
| Submission date |
Sep 02, 2023 |
| Last update date |
Oct 23, 2023 |
| Contact name |
Jessica Jacobs-Li |
| E-mail(s) |
jjacobsl@caltech.edu
|
| Organization name |
California Institute of Technology
|
| Department |
Biology
|
| Lab |
Bronner
|
| Street address |
1200 E. California Blvd
|
| City |
Pasadena |
| State/province |
California |
| ZIP/Postal code |
91125 |
| Country |
USA |
| |
|
| Platform ID |
GPL26853 |
| Series (1) |
| GSE242228 |
Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system |
|
| Relations |
| BioSample |
SAMN37253141 |
| SRA |
SRX21607438 |
