Historic ASFV Sample isolated from South Africa
Virus DNA was extracted from infected macrophage cultures presenting higher than 90% CPE. The nucleus and cytoplasmic fractions were separated using the nuclear extraction kit with the viral DNA being isolated from the cytoplasmic fraction following the manufacturers protocol (Active Motif cat# 40010). ASFV infected cells were treated with the hypotonic buffer on ice until the cell membrane was dissolved. The nucleus fraction was separated by centrifugation, the cytoplasmic fraction was collected, and DNA extracted adding 10% (v/v) of 3M Na0Ac (Sigma- Aldrich 71196 ) and an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) with a pH of 6.5-6.9 ( sigma-Aldrich P3803-100ML). The preparation was centrifuged for max speed in a tabletop centrifuge, the aqueous layer was then ethanol precipitated using 2 volumes of 100% ethanol, washed with the same volume of 70% ethanol, and dried. The resulting DNA pellet was then reconstituted in sterile water. We then used this DNA library for NGS sequencing using Nextera XT kit in the NextSeq (Illumnia, San Diego,CA) following the manufacturers protocol.