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| Status |
Public on Sep 18, 2024 |
| Title |
ATAC CFTRKO knockout, donor1 |
| Sample type |
SRA |
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| Source name |
Polarized pancreatic ductal epithelia
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| Organism |
Mustela putorius furo |
| Characteristics |
tissue: Polarized pancreatic ductal epithelia
cell type: Ductal
genotype: CFTR KO/KO
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| 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fully differentiated WT and CF ferret PDE cultures grown at ALI for 14 days were dissociated using Accutase. Cells were dissociated using Accutase and ~50,000 cells from each PDE culture were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40: Sigma). Transposition was performed using 25 μl tagmentation reaction mix from Tagment DNA kit (Illumina, CA, USA). Tagged DNA was amplified indexed, using the NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs, MA, USA) and with Nextera DNA CD Indexes (Illumina, CA, USA), using the following settings: 72°C for 5 minutes; 98°C for 30 seconds; 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. The indexed library was purified with 1.8 times the volume of Ampure XP beads (Beckman Coulter, CA, USA). Library quality was assessed using a BioAnalyzer 2100 High Sensitivity DNA Chip (Agilent Technologies, MA, USA). All DNA libraries that exhibited the correct nucleosome pattern were pooled and processed for 150bp paired-end sequencing using a HiSeq4000 (Illumina) in the University of Iowa Genomics Division.
Cells were dissociated using Accutase and ~50,000 cells from each PDE culture were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40: Sigma). Transposition was performed using 25 μl tagmentation reaction mix from Tagment DNA kit (Illumina, CA, USA). Tagged DNA was amplified indexed, using the NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs, MA, USA) and with Nextera DNA CD Indexes (Illumina, CA, USA), using the following settings: 72°C for 5 minutes; 98°C for 30 seconds; 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. The indexed library was purified with 1.8 times the volume of Ampure XP beads (Beckman Coulter, CA, USA).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Illumina
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| Data processing |
Sequences were aligned using Bowtie. Peaks were identified using MACS on Galaxy tools. Differential peak analysis was done using Diffbound on Galaxy tools.
Assembly: Mustela purorius furo, assembly ASM1176430v1.1
Supplementary files format and content: BED files
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| Submission date |
Apr 15, 2024 |
| Last update date |
Sep 18, 2024 |
| Contact name |
Kristen Wells |
| E-mail(s) |
kristen.wells-wrasman@cuanschutz.edu
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| Organization name |
University of Colorado Anschutz
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| Department |
Barbara Davis Center
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| Lab |
Barbara Davis Center
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| Street address |
1775 Aurora Ct,
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL34388 |
| Series (1) |
| GSE264021 |
CFTR Represses a PDX1 Axis to Govern Pancreatic Ductal Cell Fate [Bulk ATAC-seq] |
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| Relations |
| BioSample |
SAMN40973237 |
| SRA |
SRX24264409 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8208874_CF1_peaks_sort.bed.gz |
2.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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