GSM4705319: Biliary epithelial cells - HL6 CD133+; Homo sapiens; RNA-Seq
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, 1 ng of each total RNA sample was fragmented to a size appropriate for sequencing based on the level of degradation assessed using Bioanalyser (Agilent), and cDNA was generated using the SMARTer Stranded Total RNA-Seq Kit- Pico Input Mammalian (Illumina). 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). Libraries were multiplexed in two equimolar pools and sequenced on two flow cells on an Illumina NextSeq 550 sequencer (Illumina).