GSM8184256: MCL-19079-1; Mus musculus; Bisulfite-Seq
RNA were isolated from liver of HBV transgenic mice using the protocol as described by: Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162: 156–159. doi:10.1006/abio.1987.9999. Bisulfite treatment of protein-free genomic DNA for methylation analysis was performed using the EZ DNA Methylation-Lightning Kit (D5030; Zymo Research, Inc., Irvine, CA, USA) according to the manufacturer's instructions.  The three major viral amplicons covering 62 of the 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.  Preparation of DNA for high-throughput amplicon sequencing was performed in two PCR steps in a protocol termed targeted amplicon sequencing (TAS)  The primer pairs targeting the bisulfite converted HBV DNA were (i) 5'-ACACTGACGACATGGTTCTACACCCCCACTAACTAAAAC-3' (oligo CS1FP2; HBV nucleotide coordinates 1198-1214) and 5'- TACGGTAGCAGAGACTTGGTCTGGGTAATATTTGGTGG-3' (oligo CS2RP2; HBV nucleotide coordinates 1645-1630), (ii) 5'-ACACTGACGACATGGTTCTACAAGTTATAGAGTATTTGGTGT-3' (oligo CS1FP5a; HBV nucleotide coordinates 2244-2263) and 5'-TACGGTAGCAGAGACTTGGTCTCCCAATAAAATTCCCCA-3' (oligo CS2RP5a; HBV nucleotide coordinates 2491-2475), and (iii) 5'-ACACTGACGACATGGTTCTACAACCTCCAATCACTCAC-3' (oligo CS1FP9; HBV nucleotide coordinates 325-340) and 5'-TACGGTAGCAGAGACTTGGTCTGTTAAATAGTGGGGGAAAG-3' (oligo CS2RP9; HBV nucleotide coordinates 730-712).