 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 06, 2023 |
| Title |
PDO_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
pancreatic duct organoids
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: pancreatic duct
|
| Treatment protocol |
The extrahepatic bile duct and pancreatic duct epithelia obtained by separation and digestion were directly resuspended in Trizol, and the organoids used for sequencing and their replicates were cultured for 3-4 passages.
|
| Growth protocol |
Mouse extrahepatic bile duct and pancreatic duct were obtained from C57BL/6 mouse by direct dissection under a dissecting microscope, and then were digested at 37 ℃ with collagenase IV for 20 min after mechanical mincing, washed and centrifuged to resuspend the cells in Matrigel , and 600 μL mouse expansion media was added to 24 wells. All cells were cultured in a humidified incubator with 95% air and 5% CO2 at 37°C. The main components of mouse expansion media included D/F12 basal media, 1% Glutamax, 1% penicillin-streptomycin , 2% B27 Supplement (minus VA), 30% Wnt3A condition media, 1 mM N-acetylcysteine , 50 ng/mL EGF , 100 ng/mL R-Spondin-1, 25 ng/mL Noggin , 100 ng/mL FGF10 , 10 mM Nicotinamide and 10 μM Y27632. When the luminal organoid obtained, the culture media was changed into mouse expansion mediawithout Wnt3A and Y27632.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using Trizol reagent.
A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair,A-tailing and adapter added were implemented. The aimed products were retrieved and PCR was performed, then the library was completed.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
RNA concentration of library was measured using Qubit® RNA Assay Kit in Qubit® 3.0 to preliminary quantify and then dilute to 1ng/μl. Insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and qualified insert size was accurate quantification using StepOnePlus™ Real-Time PCR System (Library valid concentration＞10 nM).
The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated.
The reference genomes and the annotation file were downloaded from ENSEMBL database (http://www.ensembl.org/index.html). Bowtie2 v2.2.3 was used for building the genome index, and Clean Data was then aligned to the reference genome using HISAT2 v2.1.0. HISAT2 is the successor to TopHat2, which uses a modified BWT algorithm to convert reference genomes to index for faster speed and fewer resources.
Reads Count for each gene in each sample was counted by HTSeq v0.6.0, and FPKM (Fragments Per Kilobase Millon Mapped Reads) was then calculated to estimate the expression level of genes in each sample. the formula is shown as：FPKM=F*106/NL/103 F is the number of fragments in a certain sample that is assigned to a certain gene, N is the total number of mapped reads in the certain sample and L is the length of the certain gene.
Genome_build: http://www.ensembl.org/index.html
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
|
| |
|
| Submission date |
Feb 08, 2022 |
| Last update date |
Jun 06, 2023 |
| Contact name |
Miao Liu |
| E-mail(s) |
miao719@hotmail.com
|
| Organization name |
Northeast Forestry University
|
| Street address |
College of Life Sciences, Northeast Forestry University, No. 26, Hexing Road, Xiangfang District,
|
| City |
Harbin |
| ZIP/Postal code |
150040 |
| Country |
China |
| |
|
| Platform ID |
GPL21273 |
| Series (1) |
| GSE196394 |
Comparison of gene expression similarity between mouse extrahepatic bile duct and pancreatic duct epithelia and organoids prepared from these epithelia. |
|
| Relations |
| BioSample |
SAMN25759926 |
| SRA |
SRX14108327 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
