RNA-seq
Total RNA was extracted from paired normal and cancer tissues from five BTC patients using RNeasy Miniprep kits (Qiagen, Valencia, CA, USA). Subsequently complementary DNA (cDNA) was synthesized and subjected to end-repair and poly (A) addition and ligated with sequencing adapters using the TruSeq RNA Sample Prep Kit (Illumina, CA). Subsequent libraries were sequenced with an Illumina HiSeq2500 sequencer (Illumina, CA) as recommended by the manufacturer. We generated, on an average, ~43 million paired-end (2  100-bp) reads per sample.