JEG-3 cells (~50 million cells per experiment) were inoculated with ZIKV isolate PE243, at MOI: 2 TCID50/cell. 20 hours post inoculation cells were washed 3 times in PBS and were incubated for 20 minutes with 0.4 mg/ml Psoralen-triethylene glycol azide (psoralen-TEG azide, Berry & Associates) dissolved in PBS and diluted in OptiMEM I with no phenol-red (Gibco). Cells were irradiated on ice with 365 nM UV for 10 minutes using a CL-1000 crosslinker (UVP). Prolonged UVA irradiation should be avoided as it might decompose the azide moiety. Cells were lysed using RNeasy lysis buffer. Proteins were degraded by proteinase K (NEB) and RNA was purified using RNeasy midi kit (Qiagen).
Optimem 1 with no phenol red
Manufactured by Thermo Fisher Scientific
OptiMEM I with no phenol-red is a cell culture medium designed for the growth and maintenance of various cell types. It is formulated without the addition of phenol-red, a pH indicator dye. The core function of this product is to provide a balanced salt solution and nutrients required for cell growth and proliferation, while avoiding potential interference from phenol-red.
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3 protocols using optimem 1 with no phenol red
1
ZIKV Infection Profiling in JEG-3 Cells
2
Preparing SARS-CoV-2 and MERS-CoV Samples for Analysis
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Infection experiments were performed under biosafety level 3 conditions. Independent biological replicates were performed using 90-120 million cells each. Titration of virus stocks was conducted using Vero E6 cells. For SARS-CoV-2 infection, VeroE6/TMPRSS2 cells (PMID: 32165541 ) were inoculated with SARS-CoV-2 strain München-1.2/2020/984 (Rothe et al., 2020 (link)) at MOI = 2 pfu/cell for 20 h. For MERS infection, HuH7 cells were inoculated with MERS-CoV strain EMC/2012 (GenBank: JX869059.2 ; PMID: 23170002 ) (van Boheemen et al., 2012 ) at MOI = 2 pfu/cell for 20 h. Following inoculation, cells were washed 3 times with PBS and were incubated for 20 min with 0.4 mg/ml Psoralen-triethylene glycol azide (psoralen-TEG azide, Berry & Associates) diluted in PBS and supplemented with OptiMEM I with no phenol-red (GIBCO). Cells were subsequently irradiated on ice with 50 KJ/m2 365 nm UVA using a CL-1000 crosslinker (UVP). Cell lysis was performed by RNeasy lysis buffer (QIAGEN) supplemented with DTT. Proteins were degraded using proteinase K (NEB) and RNA was purified using RNeasy maxi kit (QIAGEN).
3
ZIKV Infection Profiling in JEG-3 Cells
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