Complementary DNA (cDNA) was then synthesized from the total RNA with random primers by using Superscript III Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad, CA). The sequence of nucleotides 3429−9727 of the HCV genotype 1b replicon (R6NRz) genome or nucleotides 325−9381 of the HCV genotype 1a (HCG9) genome, including all of the HCV protein coding sequence, was divided into several

segments of 1.5−3 kb with overlapping regions. Four segments of the genotype 1b replicon genome were amplified from the cDNA by polymerase chain reaction (PCR) with specific primers (Supplementary Table 2), and 7 segments of the genotype 1a (HCG9) genome were amplified from the cDNA by nested PCR with the indicated primers GDC-0199 purchase (Supplementary Table 3) by using PrimeSTAR find more GXL DNA Polymerase (TaKaRa Bio, Shiga, Japan). The amplified segments of HCV cDNA were purified from 1% agarose gels by using a MinElute Gel Extraction Kit (Qiagen, Valencia, CA) and quantified by measuring absorbance at 260 nm with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). The cDNA segments covering the coding sequence of HCV were then pooled together at approximately equimolar ratios. The Covaris S220 system (Covaris, Woburn, MA) was used to shear 500 ng

of the pooled cDNA into 700- to 800-bp fragments. The sheared cDNA fragments were purified with the MinElute PCR Purification Kit (Qiagen), ligated with RL MID adaptors (Roche Diagnostics) Non-specific serine/threonine protein kinase to prepare the multiple cDNA libraries, and further purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). The quality and quantity of the libraries were assessed by using an Agilent 2100 Bioanalyzer (Agilent

Technologies, Santa Clara, CA) and the KAPA Library Quantification Kit (Nippon Genetics, Tokyo, Japan), respectively. The libraries were then subjected to emulsion PCR, and enriched DNA beads (approximately 10% recovery) were loaded onto a picotiter plate and pyrosequenced with a GS Junior sequencer by using titanium chemistry (Roche Diagnostics). Several libraries derived from the HCV genomes generated by different treatments were sequenced in a single GS Junior run. The data obtained were analyzed by the GS Reference Mapper software (Roche Diagnostics) to identify resistant mutations. Crude extracts of the HCV subgenomic replicon cell line FLR3-112 (genotype 1b, Con-1) were used as a source of SPT in this assay. Briefly, FLR3-1 cells were suspended in HSS buffer (10 mM HEPES-KOH, 25 mM sucrose, and 0.1% sucrose monolaurate) containing 1/100 volume of protease inhibitor cocktail (Sigma, St Louis, MO) and sonicated 10 times with short pulses. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was stored at −80°C until use. Crude extract of FLR3-1 cells was added to 0.