How human cells become Zikavirus factories ljiresearch NatureComms
On day 7 of differentiation, moDCs were infected with ZIKV SD001, ZIKV PRVABC59, ZIKV-EGFP-BeH819015, or DENV UIS353 at MOIs indicated in the text. moDCs were mixed with virus, incubated for 2 h at 37 °C, washed with phosphate-buffered saline , and then resuspended in fresh medium and incubated for the indicated times at 37 °C. NPCs were infected as in.
Separation of virally infected and uninfected cells by cell sorting for RNA-seq, csRNA-seq, and qRT-PCR moDCs were infected with ZIKV or DENV2 for 24 h at MOIs of 0.5 and 1, respectively. Huh7.5 cells and NPCs were infected with ZIKV for 24 h at an MOI of 1. The cells were then collected, incubated with Zombie Violet™ Fixable Viability stain for 20 min at 4 °C, washed, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin in the presence of RNasin ribonuclease inhibitor .
cells were resuspended on ice in 130 µl RLNR1 lysis buffer , and transferred to microtubes with an AFA Fiber . All subsequent steps were performed at 4 °C. Chromatin was sheared by sonication in a 96 Place microTUBE Rack using a Covaris E220 focused-ultrasonicator for 20 cycles with the following settings: time, 60 s; duty, 5; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were recovered and spun at maximum speed for 10 min and the pellet was discarded.
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