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Sample GSM8208870

Query DataSets for GSM8208870

Status

Public on Sep 18, 2024

Title

WT, donor2

Sample type

SRA

Source name

Polarized pancreatic ductal epithelia

Organism

Mustela putorius furo

Characteristics

tissue: Polarized pancreatic ductal epithelia
cell type: Ductal
genotype: WT

Growth protocol

Pancreata were removed from 1-3 day old newborn WT and CF ferrets and digested in 5 mg/ml collagenase for 20 min at 37oC. The digested pancreas was incubated overnight in PneumaCult™-Ex Plus medium (STEMCELL Technologies, MA, USA) on 804G-coated culture dishes overnight at 37oC in a 5% CO2 incubator. The 804G coating procedure is as previously described for airway basal cells {Mou, 2016 #413}. Ductal structures that adhered to the plate were aspirated on the following day and cultured on fresh 804G-coated culture dishes in PneumaCult™-Ex Plus medium until near confluence. These cells were then passaged by incubating with Accutase (STEMCELL Technologies, MA, USA) for 5 min at 37oC. The cells were passaged continuously for 10 passages to eliminate contaminating cells and obtain a morphologically homogenous population of duct cells. Passage-10 cells were harvested using Accutase and transferred to 804G-coated transwell inserts (Corning, NY, USA) at a density of 100,000 cells per well. Following seeding, transwells were cultured in PneumaCult™-Ex Plus medium on both apical and basolateral chambers for 3 days. The medium in both the apical and basolateral chamber was then switched to PneumaCult™-ALI (STEMCELL Technologies, MA, USA) for 1 day. Air liquid interface (ALI) was established the following day by aspirating the medium from the apical chamber. Cultures were maintained at an ALI for 2 weeks before use in experiments at which time transepithelial resistance should be greater than 1000 ohms.

Extracted molecule

polyA RNA

Extraction protocol

Fully differentiated WT and CF ferret PDE cultures grown at ALI for 14 days were dissociated using Accutase followed by DNase treatment. Qiagen RNEasy mini kit was used to extract RNA from cells. Two transwells per donor were combined. RNA samples with RIN>9 were used for library prep and sequencing.
Sequencing libraries were generated by following 10X Genomics recommendations (Document CG000315). Briefly, single cells and reverse transcription master mix were partitioned into Gel Beads in partitioning oil in the 10X Chromium controller. After reverse transcription, cDNA libraries were amplified and fragmentated, followed by adaptor ligation 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

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Illumina

Data processing

Sequences from bulk RNA-seq were aligned using RSEM, using the ferret ASM1176430v1.1 genome and gtf file. Differential gene expression analysis was done using PyMINR.
Assembly: Mustela purorius furo, assembly ASM1176430v1.1
Supplementary files format and content: Tab separated values, comma separated values, matrix files

Submission date

Apr 15, 2024

Last update date

Sep 18, 2024

Contact name

Kristen Wells

E-mail(s)

[email protected]

Organization name

University of Colorado Anschutz

Department

Barbara Davis Center