Iodixanol

See Supplementary
Protocol 2 for a detailed protocol. This protocol is highly similar
to the INTACT method^{19 (link)} and
either protocol can be used for the isolation of nuclei with equivalent results.
All of the steps were carried out at 4 °C. A frozen tissue fragment ~20
mg was placed into a pre-chilled 2-ml Dounce homogenizer containing 2 ml of cold
1× homogenization buffer (320 mM sucrose, 0.1 mM EDTA, 0.1%
NP40, 5 mM CaCl₂, 3 mM Mg(Ac)₂, 10 mM Tris pH 7.8,
1× protease inhibitors (Roche, cOmplete), and 167 μM
β-mercaptoethanol, in water). Tissue was homogenized with approximately
ten strokes with the loose ‘A’ pestle, followed by 20 strokes
with the tight ‘B’ pestle. Connective tissue and residual debris
were precleared by filtration through an 80-μm nylon mesh filter
followed by centrifugation for 1 min at 100 r.c.f. While avoiding the pelleted
debris, 400 μl was transferred to a pre-chilled 2-ml round bottom
Lo-Bind Eppendorf tube. An equal volume (400 μl) of a 50%
iodixanol solution (50% iodixanol in 1× homogenization buffer)
was added and mixed by pipetting to make a final concentration of 25%
iodixanol. 600 μl of a 29% iodixanol solution (29%
iodixanol in 1× homogenization buffer containing 480 mM sucrose) was
layered underneath the 25% iodixanol mixture. A clearly defined
interface should be visible. In a similar fashion, 600 μl of a
35% iodixanol solution (35% iodixanol in 1×
homogenization containing 480 mM sucrose) was layered underneath the 29%
iodixanol solution. Again, a clearly defined interface should be visible between
all three layers. In a swinging-bucket centrifuge, nuclei were centrifuged for
20 min at 3,000 r.c.f. After centrifugation, the nuclei were present at the
interface of the 29% and 35% iodixanol solutions. This band with
the nuclei was collected in a 300 μl volume and transferred to a
pre-chilled tube. Nuclei were counted after addition of trypan blue, which
stains all nuclei due to membrane permeabilization from freezing. 50,000 counted
nuclei were then transferred to a tube containing 1 ml of ATAC-seq RSB with
0.1% Tween-20. Nuclei were pelleted by centrifugation at 500 r.c.f. for
10 min in a pre-chilled (4 °C) fixed-angle centrifuge. Supernatant was
removed using the two pipetting steps described above. Because the nuclei were
already permeabilized, no lysis step was performed, and the transposition mix
(25 μl 2× TD buffer, 2.5 μl transposase (100 nM final),
16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl
10% Tween-20, 5 μl water) was added directly to the nuclear
pellet and mixed by pipetting up and down six times. Transposition reactions
were incubated at 37 °C for 30 min in a thermomixer with shaking at
1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5
columns. The remainder of the ATAC-seq library preparation was performed as
described previously¹⁸ .

Noncytopathic, cell culture-adapted HM175/p16 HAV^{9 (link)} was propagated in Huh-7.5 cells. Reverse molecular genetics studies were carried out with pHM175/18f, a molecular clone of a related cytopathic variant^{28 (link)}. Buoyant density was assessed in 8–40% iodixanol (Opti-Prep) gradients centrifuged at 141,000 × g for 48 hrs at 4 °C. Viral RNA was measured by qRT-PCR with primers targeting the 5’-untranslated region. Infectivity was quantified by infrared fluorescence immunofocus assay (IR-FIFA)^{10 (link)}. For RNAi studies, cells were transfected with SmartPool siRNAs (Dharmacon) and samples collected 48–72 hrs later for viral RNA quantification. To analyze VP2-ALIX interactions, cell lysates were prepared 48 hrs after electroporation of mutant and wild-type viral RNAs, treated with RNase, and immunoprecipitated. RNA extracted from immunoprecipitates was assayed by HAV-specific qRT-PCR. For intracellular neutralization, cells were incubated with eHAV for 1 hr at 37 °C, then washed extensively. Antibodies were added at intervals, and intra- and extracellular HAV RNA quantified at 48–72 hrs. For standard neutralization assays, virus was incubated with antibodies for 1 hr at 37 °C, then inoculated onto cells.