An encapsulated lymphatic organ through which venous blood filters.
Attributes by original data submitter
Sample
source_name
HBV transgenic mouse lineage 1.3.32
genetic background
SV129
genotype
HBV (wild type)
Sex
male
age
Adult
organ
Spleen
Sequenced DNA Library
library_strategy
Bisulfite-Seq
library_source
GENOMIC
library_selection
RANDOM
library_construction_protocol
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 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)” as described previously (50,51). Thirteen HBV primer pairs targeting 95 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’-ACACTGACGACATGGTTCTACAGGGTGGGTGTTAATTTG-3’ (oligo CS1FP5; HBV nucleotide coordinates 2114-2130) and 5’-TACGGTAGCAGAGACTTGGTCTCCAAAAAATACTAACATTAAAAT-3’ (oligo CS2RP5; HBV nucleotide coordinates 2464-2442), (vii) 5’- ACACTGACGACATGGTTCTACAAGTTATAGAGTATTTGGTGT-3’ (oligo CS1FP5a; HBV nucleotide coordinates 2244-2263) and 5’-TACGGTAGCAGAGACTTGGTCTCCCAATAAAATTCCCCA-3’ (oligo CS2RP5a; HBV nucleotide coordinates 2491-2475), (viii) 5’- ACACTGACGACATGGTTCTACAGATTGTAATTGATTATGTTTG -3’ (oligo CS1FP6; HBV nucleotide coordinates 2625-2645) and 5’- TACGGTAGCAGAGACTTGGTCTCCATACTATAAATCTTATTCCC-3’ (oligo CS2RP6; HBV nucleotide coordinates 2832-2853), (ix) 5’- ACACTGACGACATGGTTCTACAGGGAATAAGATTTATAGTATGG (oligo CS1FP7; HBV nucleotide coordinates 2832-2853) and 5’-TACGGTAGCAGAGACTTGGTCTTAAACCTAAAAACTCCACC-3’ (oligo CS2RP7; HBV nucleotide coordinates 3061-3043, (x) 5’-ACACTGACGACATGGTTCTACAAATCAAAAAAACAACCTACC-3’ (oligo CS1FP8; HBV nucleotide coordinates 3115-3134) and 5’-TACGGTAGCAGAGACTTGGTCTGTGAGTGATTGGAGGT-3’ (oligo CS2RP8; HBV nucleotide coordinates 340-325), (xi) 5’-ACACTGACGACATGGTTCTACAACCTCCAATCACTCAC-3’ (oligo CS1FP9; HBV nucleotide coordinates 325-340) and 5’-TACGGTAGCAGAGACTTGGTCTGTTAAATAGTGGGGGAAAG-3’ (oligo CS2RP9; HBV nucleotide coordinates 730-712), (xii) 5’-ACACTGACGACATGGTTCTACAGGATGATGTGGTATTGG-3’ (oligo CS1FP10; HBV nucleotide coordinates 743-759) and 5’-TACGGTAGCAGAGACTTGGTCTCAAAACCCAAAAAACCCAC-3’ (oligo CS2RP10; HBV nucleotide coordinates 1020-1002), and (xiii) 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, 72°C for 60 s. Finally, a seven 7 minute incubation at 72°C was performed. The HBV amplicons were subsequently subjected to a second stage PCR reaction (AccuPrime SuperMix II, Life Technologies), 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 unique 10 base sample-specific barcodes are indicated with bold Ns and represent a subset of 384 unique sequences (Fluidigm). PCR amplification involved 5 minutes denaturation at 95°C, followed by 8 cycles of 95°C for 30 s, 60°C for 30 s, 68°C for 30 s. Finally, a seven minute incubation at 68°C was performed. Pooled libraries were sequenced using an Illumina MiSeq instrument and data were analyzed using the Casava1.8 pipeline. Sequencing was performed using MiSeq V2 chemistry, with paired-end 2x300 base reads, employing Fluidigm custom sequencing primers. Sequence data were demultiplexed on instrument.