title: GSM1548447: ICC17 (Exome-seq); Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted from homogenized cholangiocarcinoma samples and their normal conterparts usingTRizol Regent (Invitrogen). Quantity of RNA was measured using Quant-iT Ribogreen RNA assay kit and quality was assessed using a Bioanalyzer. One µg of RNA was used for subsequent RNA-seq library generation. DNA was isolated using the ChargeSwitch® gDNA Mini Tissue Ki/t (Invitrogen) after tissue disruption with a homogenizer. DNA quantity was assessed through Quant-It PicoGreen dsDNA Assay kit (Invitrogen) and its integrity confirmed by agarose gel. For RNA-seq, the sequencing library was prepared with the standard TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). Briefly, total RNA was poly-A-selected and then fragmented. The cDNA was synthesized using random hexamers, end-repaired and ligated with appropriate adaptors for sequencing. The library then underwent size selection and purification using AMPure XP beads (Beckman Coulter, CA, USA). The appropriate Illumina recommended 6 bp barcode bases are introduced at one end of the adaptors during PCR amplification step. The size and concentration of the RNAseq libraries was measured by Bioanalyzer and Qubit fluorometry (Life Technologies, NY, USA) before loading onto the sequencer. The mRNA libraries were sequenced on the Illumina HiSeq 2500 System with 100 nucleotide single-end reads, according to the standard manufacturer's protocol (Illumina, CA, USA). For exome-seq, total genomic DNA library is generated following the manufacturer protocol (NEBNext DNA Library Prep Master Mix Set for Illumina, New England Biolabs, Ipswich, MA). For whole exome sequencing (WES), the library then undergoes solution-based hybridization to an oligonucleotide pool designed to enrich for the whole exome regions of interests. The library is captured by following the manufacturer protocol (SeqCap EZ Human Exome Library v3.0 User Guide. Roche NimbleGen. Madison, WI).