title: Molecular Profiling of Gallbladder Cancer (MPOG): Sample GBC_SAMPLE_001_1069682
design: Quality control was performed on total RNA using the Qubit BR RNA assay (ThermoFisher Scientific) and Agilent RNA 6000 Pico Kit (Agilent Technologies). 100-500ng of total RNA in 10ul was used as input. Libraries were prepared using the Kapa RNA HyperPrep Kit with RiboErase per manufacturer's instructions with slight modifications (Kapa Biosystems, part# KK8561). Briefly, following depletion of ribosomal RNA, fragmentation was performed at 85C for 4.5 minutes for good quality samples, 85C for 4 minutes for average quality, and 65C for 1-1.5 min for the lowest quality and/or lowest input samples. Following 1st and 2nd strand cDNA synthesis, 1.5uM NEXTflex-96 DNA Barcodes (BioO Scientific) were ligated to the A-tailed cDNA. Eight cycles of library amplification were performed, followed by two rounds of 1X Ampure purification. Final libraries were quantified by Qubit, equal molar pooled at 16 samples per pool and sequenced on the Illumina HiSeq 2500 using the SBS 2x125 bp and Cluster v4 kits.