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Sample GSM4705322

Query DataSets for GSM4705322

Status

Public on Mar 03, 2022

Title

Biliary epithelial cells - HL9 CD133-

Sample type

SRA

Source name

biliary epithelium

Organism

Homo sapiens

Characteristics

cell type: CD133- biliary epithelial cells
disease state: steatotic
individual: HL9

Growth protocol

Sorted hBEC were grown as organoids by plating onto low-adherence plates (Greiner) in hemispheres of growth factor reduced Matrigel (Corning) and grown in hBEC Expansion Medium: Advanced DMEMF12 +1%P/S, 1% Glut containing 10%FCS, 10 mM HEPES pH7, 10 mM Nicotinamide, 1x N2 Supplement, 1x B27 Supplement, 50 ng/ml EGF, 10 nM Gastrin, 100 ng/ml FGF-10, 25 ng/ml HGF, 500 ng/ml R-Spondin 1, 1.25 mM NAC, 5 uM A-83-01 and 10 uM Forskolin. For the first three days of culture medium was supplemented with 100 ng/ml Noggin, 100 ng/ml Wnt and 10 uM Y27632. Medium was changed every three days and cells passaged once confluent every 7 to 10 days. Cells were routinely tested for mycoplasma contamination using MycoAlert Assay Control set (Lonza).

Extracted molecule

total RNA

Extraction protocol

Cryopreserved sections (or fresh samples of tissue) were defrosted at room temperature and digested for 30 min at 37°C in a solution of Collagenase (2 ug/ml, Gibco)-Dispase (1 ug/ml, Gibco)-Bovine Fetal Calf Serum (2% FCS, Gibco) in Advanced DMEMF12 (LifeTech) supplemented with 1% Penicillin/Streptomycin (P/S, Gibco) and 1% L-Glutamine (Glut, Gibco). When the intrahepatic bile ducts could be observed by microscope inspection the sample was centrifuged at 100g for 5 mins, resuspended in Versene (Thermo Fisher) and incubated at 37°C for 1 hour. Dissociated cells were filtered through a 40 um cell strainer and washed in Advanced DMEMF12. Cells were resuspended in Advanced DMEMF12 underlayered with an equal volume of 20% and 50% (v/v) Percoll (Sigma). Following centrifugation at 1800g for 30 mins at 4°C, the HPC rich fraction lying between the 20 and the 50% Percoll layers was collected, washed and re-suspended in PBS with 2% FCS and 100 mmol EDTA (Sigma) for FACS staining procedure. Cells were incubated for 1 hour with EpCAM-BV650 conjugated antibody, CD24-BV421, CD133-APC, CD45-488 and CD31-488 at the concentrations stated in Table 1. Samples were stained with SYTOX-AADvanced dead cell stain kit (ThermoFisher) prior analysis in a FACSAria Fusion (BD Biosciences). Trigger pulse width was used to exclude cell doublets from analysis and collection.
For library preparation, 1ng of each total RNA sample was fragmented to a size appropriate for sequencing based on the level of degradation assessed using an Agilent Bioanalyser (Agilent), and cDNA was generated using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio, # 634411). AMPure XP beads (Beckman Coulter) were then used to purify the cDNA library. Depletion of ribosomal cDNA was achieved using ZapR and R-probes. The library fragments originating from rRNA (18S and 28S) and mitochondrial rRNA (m12S and m16S) were cut by ZapR in the presence of R-probes (mammalian-specific). R-probes were hybridized to ribosomal RNA and mitochondrial rRNA sequences derived from the human mitochondrial genome and are therefore strictly human specific. Uncleaved fragments were then enriched by 15 cycles of PCR before a final purification using AMPure XP beads (Beckman Coulter). Completed libraries were quantified using the Qubit 2.0 Fluorometer and the Qubit dsDNA HS assay and assessed for quality and size distribution of library fragments using the Agilent Bioanalyser and the DNA HS kit (Agilent).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

HL9minus
Biliary epithelial cells of biliary origin.
hBEC_gene_counts_matrix.txt

Data processing

Base call data generated by Illumina NextSeq 550, NextSeq 2000
Read quality assessed using FastQC (v.0.11.9) and MultiQC (v.1.3.dev0).
Adapter and poly-G trimming with Cutadapt (v.1.16).
Read alignment with STAR (v.2.7.1a).
Read counting with featureCounts (Rsubread Bioconductor package v.2.0.1).
Differential expression computed using DESeq2 (v.1.26.0).
Genome_build: GRCh38.99
Supplementary_files_format_and_content: Tab separated matrix tables with raw gene counts for all genes and every sample

Submission date

Jul 31, 2020

Last update date

Mar 03, 2022

Contact name

Stuart Forbes

Organization name

The University of Edinburgh

Department

Centre for Regenerative Medicine

Street address

5 Little France Drive