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Sample GSM7431965 Query DataSets for GSM7431965
Status Public on Aug 01, 2023
Title FOXI1-CreERT2::ROSA-TG ferret airway epithelial cells - Donor 11
Sample type SRA
 
Source name Ferret airway epithelial cells cultured in air-liquid interface (ALI)
Organism Mustela putorius furo
Characteristics strain: None
treatment: None
time point: None
genotype: FOXI1-CreERT2::ROSA-TG
cell type: airway epithelial cells
donor: Donor 11
Treatment protocol For scRNA-seq experiments, cultures were treated with OH-Tam during differentiation at ALI from day-1 to day-17 (media change every other day) and cells were harvested at 21 days of ALI. This was done since it gave rise to a greater number of EGFP+ cells in the cultures following full differentiation.
Growth protocol Ferret tracheal airway basal stem cell were isolated using an enzymatic digestion method similar to previous reports (Lynch, T. J. et al, Stem Cells 2016; 34, 2758-2771). The cells were cultured in PneumaCult-Ex Plus media (Stem Cell Technologies) on plastic plates pre-coated with laminin enriched 804G-conditioned medium. For passaging, the cells were detached with Accutase (Stem Cell Technologies) and re-seeded at a 1:4 split on 804G coated plates as previously described52. For differentiation at ALI, ferret basal cells were seeded onto transwell membranes coated with 804G in PneumaCult-Ex Plus media for 24 hrs and then lifted to an ALI with PneumaCult-ALI medium (Stem Cell Technologies) placed only on the basal side of the transwell.
Extracted molecule total RNA
Extraction protocol Fully differentiated ferret airway epithelia ALI cultures were dissociated using Accumax (Stem Cell Technologies) followed by DNase treatment. Cells were filtered through a 20 ╬╝Mstrainer and pelleted in 0.04% BSA PBS at 500g for 10 min. Nonviable dead cells were removedby using MACS Dead Cell Removal Kit following 10X Genomics recommendations (DocumentCG00039).
Single cells were counted on a Thermo countess cell counter and 0.04% BSA/PBSwas added to achieve a targeted concentration of 1000 cells/╬╝L. Ionocyte-enrichment wasperformed on OH-Tam treated FOXI1-CreERT2::ROSA-TG ALI cultures followed by FACSisolation of EGFP and Tomato positive cells. FACS isolated EGFP positive cells where thenmixed with Tomato positive epithelial cells to achieve ~10,000 total cells for 10x sequencing.The ratio of EGFP to Tomato positive cells varied for each experiment depending on the yield oflineage-labeled cells.
Sequencing libraries were generated by following 10X Genomicsrecommendations (Document CG000315). Briefly, single cells and reverse transcription mastermix were partitioned into Gel Beads in partitioning oil in the 10X Chromium controller. Afterreverse transcription, cDNA libraries were amplified and fragmentated, followed by adaptorligation and sample index PCR reaction. Libraries were sequenced on NovaSeq 6000 platform by the University of Iowa Genomics Division.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Data processing 10X Chromium V3 Chemistry Single-cell RNA Seq
kallisto bustools pseudoalignment
log transformation and TPM normalization (Seurat R package)
PCA (Seurat R package)
UMAP and leiden clustering (Seurat R)
Assembly: https://www.ncbi.nlm.nih.gov/assembly/GCF_011764305.1
Supplementary files format and content: Log2(Transcripts per million (TPM) + 1) expression values (derived from kallisto bustools for each gene (rows) in each single-cell (columns)
 
Submission date May 29, 2023
Last update date Aug 01, 2023
Contact name Adam Haber
E-mail(s) ahaber@hsph.harvard.edu
Organization name Harvard T. H. Chan School of Public Health
Street address 665 Huntington Ave, HSPH Building 1, Room 305
City Boston
State/province Massachusetts
ZIP/Postal code 02115
Country USA
 
Platform ID GPL28369
Series (1)
GSE233654 Transgenic Ferret Models Define Pulmonary Ionocyte Diversity and Function
Relations
BioSample SAMN35522044
SRA SRX20533832

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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