title: HBV-HCC enriched single cell sequencing
design: The fresh frozen tumor tissue samples were thaw in water bath and digested into cell solution by collagenase. With sufficient collagenase dissociation and dilution, the cancer tissues were separated into single cells solution, cell clusters and cell debris. Suspension was filtered by injecting into the membrane filter to wipe out the massive cell clusters. To avoid contamination of cell debris, suspension was re-suspended and centrifuged in Phosphate Buffered Saline (PBS) for 5 times. After filtration, cell suspension was added into a PBS droplet containing 0.5% BSA. Single cell isolation was performed by micromanipulation under microscope and cells with intact cell membrane were randomly selected for single cell sequencing. For each cell, WGA was performed with MDA.  HIVID (Li et al., 2013) procedure was then used to enrich for sequences containing HBV genome sequence. DNA library from amplified single cell genome was hybridized with the biotinylated HBV probe to enrich DNA fragments containing HBV DNA sequences. Then, the enriched libraries were quantified and subjected to 101 cycles paired-end index sequencing in Illumina HiSeq 2000 sequencer according to manufacturer's instructions (Illumina Inc., San Diego, CA).