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| Status |
Public on Jan 31, 2024 |
| Title |
Foliate_Organoid_Pig5 |
| Sample type |
SRA |
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| Source name |
Foliate cell line
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| Organism |
Sus scrofa |
| Characteristics |
cell line: Foliate cell line
cell type: Stem cells/mature taste cells
treatment: LPM
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| Growth protocol |
The plated organoid cultures were fed with 2ml fresh LPM every 48hrs. LPM was made by adding the following peptides and small molecules to LGR6 base media (LBM); EGF (50 ng/ml), FGF10 (100 ng/ml), FGF2 (10 ng/ml), R-Spondin-2 (500 ng/ml), Noggin (100 ng/ml), Human SHH (100 ng/ml), Y-27632 (10µM) from Peprotech, hu-Wnt3a (100 ng/ml) from R&D Systems, Insulin (10nM) from Gibco, A-83-01 (0.5µM), SB202190 (10µM), CHIR99021 (2.5µM), Prostaglandin E2 (20nM) from BioGems, Forskolin (1µM) from Tocris, and N-Acetyl-Cysteine (1.25µM), Nicotinamide (10mM) from Sigma-Aldrich. LBM was made by adding the following to DMEM/F12 GlutaMAX from Gibco; Hepes (10mM) and B27 insulin free (2%) from Gibco, N21 Max insulin free (2%) from R&D Systems and Primocin (10µg/ml) from Invivogen
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| Extracted molecule |
total RNA |
| Extraction protocol |
Organoid harvesting from 6-well/12-well plates was done using the Cultrex™ Organoid Harvesting Solution (Cat 3700-100-01). Briefly; working on ice the cell culture media was gently aspirated and each well was washed with PBS (2ml/1ml). 1ml of the organoid harvesting solution was added to each well, the domes gently dislodged using a cell scraper/lifter and incubated for 1hr at 4C0 on a rocking shaker (Reliable Scientific Inc.) at a speed of 50, for BME digestion. Post incubation, the organoids were spun down and washed three times with 1ml PBS. Each spin down was done at 400g for 4mins at 4C0. The palleted organoids from each well were lysed in 600µl of RLT Plus lysis buffer (Qiagen) with 6µl of ß-mercaptoethanol (Sigma Life Science Cat# 63689-100ML-F) and homogenized using ~200µl of 1.0mm Zirconia (BioSpec Cat# 11079110ZX) beads with a single 30 second, 6,000 rpm cycle on a Precellys24 homogenizer (Bertin Corp). Homogenized solution was spun down for 20mins at 8000rpm prior to RNA isolation using RNEasy Plus Mini Kit (Qiagen Cat# 74134/74136) according to manufacturer’s protocol. FP were homogenized in the Extraction Buffer provided in the Arcturus™ PicoPure™ RNA Isolation Kit (Applied Biosystems Cat# 12204-01) using the same bead based method described above. RNA extraction followed the recommended Arcturus™ PicoPure™ RNA Isolation Kit protocol without performing the DNAse step since TaqMan probes are at the junctions of gene exons
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Pre-processed (post adapter and low-quality sequence removal) paired-end raw fastq sequences received from Nonogene Inc. were analyzed using FastQC 0.11.9 for quality assurance, and aligned against Sus scrofa 11.1 genome using the STAR aligner 2.7.10b in “GeneCounts” mode. Alignment quality for each sample was checked using MultiQC 1.14. Post-quality assurance, the resulting BAM files from STAR aligner were run through featureCounts in “countReadPairs” mode to obtain gene-wise transcript counts for each sample. A gene-wise count matrix was compiled for samples pertaining to each analysis (e.g Organoids Vs Foliate etc.) and subjected to subsequent differential gene expression analysis using DESeq2 available in Bioconductor version 3.17
Assembly: Sus scrofa 11.1
Supplementary files format and content: All processed data files are the default ouput of featureCounts software in txt format
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| Submission date |
Dec 29, 2023 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Josephine M. Egan |
| E-mail(s) |
eganj@grc.nia.nih.gov
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| Organization name |
National Institute on Aging - NIH
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| Department |
Laboratory of Clinical Investigation
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| Lab |
Diabetes Section
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| Street address |
BRC Suite 100, 251 Bayview Boulevard
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21224 |
| Country |
USA |
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| Platform ID |
GPL26351 |
| Series (1) |
| GSE252240 |
Pig Taste Cell-Derived Organoids Synthesize Insulin |
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| Relations |
| BioSample |
SAMN39195627 |
| SRA |
SRX23057176 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7997945_S17_featureCounts.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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