title: GSM5258520: B10485-NT3; Mus musculus; Bisulfite-Seq
library_construction_protocol: Protein-free mouse genomic DNA was isolated from liver tissue as described (McFadden, V.C., Shalaby, R.E., Iram, S., Oropeza, C.E., Landolfi, J.A., Lyubimov, A.V., Maienschein-Cline, M., Green, S.J., Kaestner, K.H., and McLachlan, A. (2017). PLOS Pathogens 13, e1006239). Genomic DNA for methylation analysis was performed using the EZ DNA Methylation-Lightning™ Kit (D5030; Zymo Research, Inc., Irvine, CA, USA). The 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 as described previously (McFadden et al. 2017).  Twelve HBV primer pairs targeting 94 of the 99 CpG sites were used.   The primer pairs targeting the bisulfite converted HBV DNA were (i) 5'-ACACTGACGACATGGTTCTACACAATACCTAAACCTTTACCC-3' (oligo CS1FP1; HBV nucleotide coordinates 1131-1150) and 5'-TACGGTAGCAGAGACTTGGTCTGTTTTAGTTAGTGGGGGT-3' (oligo CS2RP1; HBV coordinates 1214-1197), (ii) 5'-ACACTGACGACATGGTTCTACACAAACTTTCACTTTCTC-3' (oligo CS1FP1a; HBV coordinates 1086-1102) and 5'-TACGGTAGCAGAGACTTGGTCTGTTGATGGTTTATGATTAA (oligo CS2RP1a; HBV coordinates 1233-1215), (iii) 5'-ACACTGACGACATGGTTCTACACCCCCACTAACTAAAAC-3' (oligo CS1FP2; HBV nucleotide coordinates 1198-1214) and 5'-TACGGTAGCAGAGACTTGGTCTGGGTAATATTTGGTGG-3' (oligo CS2RP2; HBV nucleotide coordinates 1645-1630), (iv) 5'-ACACTGACGACATGGTTCTACATTAAACTCTCAACAATATCA-3' (oligo CS1FP3; HBV nucleotide coordinates 1668-1687) and 5'-TACGGTAGCAGAGACTTGGTCTAAGTTATTTAAGGTATAGTTTG-3' (oligoCS2RP3; HBV nucleotide coordinates 1896-1875), (v) 5'-ACACTGACGACATGGTTCTACAGTGGTTTTGGGGTATGG-3' (oligo CS1FP4; HBV nucleotide coordinates 1890-1906) and 5'-TACGGTAGCAGAGACTTGGTCTCAAATTAACACCCACCC-3' (oligo CS2RP4; HBV nucleotide coordinates 2130-2114), (vi) 5'-ACACTGACGACATGGTTCTACAAGTTATAGAGTATTTGGTGT-3' (oligo CS1FP5a; HBV nucleotide coordinates 2244-2263) and 5'-TACGGTAGCAGAGACTTGGTCTCCCAATAAAATTCCCCA-3' (oligo CS2RP5a; HBV nucleotide coordinates 2491-2475), (vii) 5'- ACACTGACGACATGGTTCTACAGATTGTAATTGATTATGTTTG -3' (oligo CS1FP6; HBV nucleotide coordinates 2625-2645) and 5'- TACGGTAGCAGAGACTTGGTCTCCATACTATAAATCTTATTCCC-3' (oligo CS2RP6; HBV nucleotide coordinates 2832-2853), (viii) 5'-ACACTGACGACATGGTTCTACAGGGAATAAGATTTATAGTATGG (oligo CS1FP7; HBV  nucleotide coordinates 2832-2853) and 5'-TACGGTAGCAGAGACTTGGTCTTAAACCTAAAAACTCCACC-3' (oligo CS2RP7; HBV nucleotide coordinates 3061-3043, (ix) 5'-ACACTGACGACATGGTTCTACAAATCAAAAAAACAACCTACC-3' (oligo CS1FP8; HBV  nucleotide coordinates 3115-3134) and 5'-TACGGTAGCAGAGACTTGGTCTGTGAGTGATTGGAGGT-3' (oligo CS2RP8; HBV  nucleotide coordinates 340-325), (x) 5'-ACACTGACGACATGGTTCTACAACCTCCAATCACTCAC-3' (oligo CS1FP9; HBV  nucleotide coordinates 325-340) and 5'-TACGGTAGCAGAGACTTGGTCTGTTAAATAGTGGGGGAAAG-3' (oligo CS2RP9; HBV nucleotide coordinates 730-712), (xi) 5'-ACACTGACGACATGGTTCTACAGGATGATGTGGTATTGG-3' (oligo CS1FP10; HBV nucleotide coordinates 743-759) and 5'-TACGGTAGCAGAGACTTGGTCTCAAAACCCAAAAAACCCAC-3' (oligo CS2RP10; HBV nucleotide coordinates 1020-1002), and (xii) 5'-  ACACTGACGACATGGTTCTACATTGATGTTTTTGTATGTA-3' (oligo CS1FP11;  HBV nucleotide coordinates 1053-1070) and 5'-TACGGTAGCAGAGACTTGGTCTATAACCAAACCCCAACC-3' (oligo CS2RP11; HBV nucleotide coordinates 1222-1206).  These primers contained linker sequences (underlined) at the 5' ends of the oligonucleotides, termed common sequences (CS1 and CS2). These PCR reactions were performed using ZymoTaq PreMix according to the manufacturer's instructions (Zymo Research).  PCR amplification involved 10 minutes denaturation at 95°C, followed by 40 cycles of 95°C for 30 s, 50°C for 40 s (ramp rate 95-50 was set to 0.3C/s), 72°C for 60 s.  Finally, a 7 minute incubation at 72°C was performed.  The HBV amplicons were subsequently subjected to a second stage PCR reaction (MyTaq HS Mix, 2X, Bioline USA Inc., Taunton, MA, USA), used to incorporate unique barcodes and sequencing adapters. The PCR primers used were 5'-  AATGATACGGCGACCACCGAGATCTACACTGACGACATGGTTCTACA-3' (oligo PE1CS1) and 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNNNTACGGTAGCAGAGACTTGGTCT-3' (oligo PE2BCCS2).  The 10-base sample-specific barcodes are indicated with bold Ns and represent barcode sequences from a set of 384 unique primer pairs (Fluidigm Access Array Barcode Library for Illumina Sequencers).  PCR amplification involved 5 minutes denaturation at 95°C, followed by 8 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s.  Finally, a 7 minute incubation at 72°C was performed.  The resulting final PCR products (libraries) contain Illumina sequencing adapters and a sample-specific barcode. Pooled libraries were sequenced using an Illumina MiSeq instrument and data were analyzed using the Casava1.8 pipeline. Sequencing was performed using MiSeq V3 chemistry, with paired-end 2x300 base reads, employing Access Array custom sequencing primers.