Pippin prep system

Cells were pre-treated with JQ1 (1 µM) or A-241 (250 nM) for 1 h prior to the addition of recombinant murine IFNy (1 ng/mL), or vehicle control, for an additional 2 h (3 h total incubation with small molecules). Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) was performed using an improved protocol to reduce mitochondria from the transposition reaction [40 (link)]]. Briefly, 5e5 MM1.S cells were cultured in duplicate with JQ1, A-485, or DMSO vehicle as described above. Cells were washed once in ice-cold PBS and lysed in ATAC lysis buffer (0.1% Tween-20, 0.1% NP-40, 3 mM MgCl₂, 10 mM NaCl, 10 mM Tris HCl pH 7.4). Tagmentation was then performed with Tn5 transposase and 2 × TD Buffer (Nextera DNA Library Prep Kit, Illumina) for 30 min at 37 °C (in a thermocycler). Tagmented DNA was immediately purified using a MinElute column (Qiagen, #28004) and then amplified for 12 cycles using 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602) and Illumina-compatible/barcoded primers. The amplified libraries were purified using MinElute columns (Qiagen) and sequenced on an Illumina NextSeq 500 with 75 b.p. single-end reads. Library QC and quantification were performed using D1000 high-sensitivity screen tape with 4200 TapeStation Instrument (Agilent Technologies), and the size was selected between 200 and 500 bp using a Pippin Prep system (Sage Science).

1 μl of each amplicon library was run on a Bioanalyzer HS DNA Chip to assess the amplicon size and quantified using a Qubit ${}^{Ⓡ}$ dsDNA HS assay. Each amplicon library was size selected according to the expected amplicon size (±50 bp) using a pre-cast 1.5 % agarose with ethidium bromide gel cassette on the Pippin Prep System (Sage Science). The library concentration for Illumina and 454 amplicon libraries was assessed using a SYBR green qPCR assay with primers specific to each platform (Kapa).

ASFV DNA was extracted from infected cells and quantified as described earlier9 (link). Full-length sequencing of the virus genome was performed as described elsewhere9 (link). Briefly, one microgram of virus DNA was enzymatically sheared and the resulting fragmented DNA size distribution was assessed. Adapters and library barcodes were ligated to the fragmented DNA. The appropriate size range of the adapter-ligated library was collected using the Pippin Prep™ system (Sage Science, Beverly, MA) followed by normalization of library concentration. The DNA library was then clonally amplified onto ISPs and enriched. Enriched template ISPs were prepared and loaded onto Ion chips for sequencing using an Ion Torrent PGM™ instrument. Sequence analysis was performed using Galaxy (https://usegalaxy.org/) and CLC Genomics Workbench (CLCBio, Waltham, MA).