Plateloc thermal microplate sealer

High-throughput real-time RT-PCR amplifications (M, β-actin, H1_av, H1_hu, H1_{huΔ146–147,} H1pdm, H3, N1, N1pdm and N2) were conducted on a LightCycler^®1536 real-time PCR system (Roche, Meylan, France). A Bravo automated liquid handling platform equipped with a chiller and a PlateLoc thermal microplate sealer (Agilent Technologies, La Jolla, CA, USA) was used as a microplate dispenser. Each RT-qPCR mixture contained 1 μL of RNA extract and 1 μL of master mix containing 1X RealTime ready DNA Probes Master (Roche, Meylan, France), 2X GoScript RT Mix for 1-step RT-qPCR (50X) (Promega, Fitchburg, WI, USA), 800 nM of each concerned primer, with the exception of the M gene forward primer that was included at a final concentration of 400 nM, and 250 nM of standard or MGB-labelled TaqMan probe. The one-step real-time RT-PCR program involved a 30 min reverse transcription of RNA at 45 °C, followed by a 2 min denaturation step at 95 °C, and lastly 40 cycles of 0 s (i.e. a pulse) at 95 °C and 1 min at 58 °C.

The 2684 SELLECK compounds were received in 96-well plates and reformatted into 1536-well flat, black-bottom polypropylene plates (Greiner Bio-One). In total, 50 nl of each compound solution was dispensed in DMSO using an automated Echo 550 acoustic liquid dispenser (Labcyte). Compounds were formatted into the assay plates, at a final concentration of 30 μM, with the first two and last two columns loaded with DMSO only (compound-free controls). These assay plates were then heat-sealed using a PlateLoc Thermal Microplate Sealer (Agilent Technologies) and stored at −20 °C. Before screening, compound plates were equilibrated to room temperature (25 °C). In total, 0.5 μM mNG-ABD1 without or with 1 μM Alexa-568-labeled actin was dispensed by a Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific) into the 1536-well assay plates containing the compounds. Plates were incubated at room temperature for 60 min before recording the data with the FLTPR. A control measurement with 0.5 μM mNG (fluorescent protein only) was performed to eliminate the compounds that affected the environment of the fluorescence protein.

qPCR analysis of both
loxTags and PCRTags was done by adapting an earlier established protocol.^{15 (link)} The methodology described here represents the
optimized procedure after the initial loxTag screen. LoxTag and PCRTag
primer pairs were dispensed using an Echo525 instrument (Labcyte).
25 nL of 50 μM premixed forward and reverse primers was dispensed
in 384 PCR plates (Sarstedt, 72.1984.202); 25 nL was not included
in the total volume calculation. Primer plates were prepared in bulk
and stored for up to 4 weeks. To perform the 1 μL qPCR assay,
1× Luna Universal qPCR Master Mix (M3003, NEB) containing 2 ng/μL
template DNA was dispensed using a nanoliter dispenser (Cobra 4-channel
dispenser, ARI). Plates were spun down at 500 × g for 5 min followed by sealing at 180 °C for 2 s with optical
clear permanent seal (Agilent, 24212-001) using the Plateloc Thermal
Microplate Sealer (Agilent). qPCRs were run using Applied Biosystems
QuantStudio 5. Samples were preincubated at 50 °C (1.6 °C/s)
for 2 min followed by 1 min 95 °C (2.57 °C/s). A two-step
PCR reaction was performed with 30 cycles each 95 °C (2.57 °C/s)
1 s and 67 °C (2 °C/s) 1 min, with single acquisition. Melting
curve was acquired from 97 °C (0.1 °C/s) with continuous
acquisition. Raw data were exported and analyzed by using a custom
R script applying ggPlot2 for heatmap generation.

Test compounds were prepared by serially diluting a 10 mM compound stock threefold with DMSO for eight concentration points in the master compound plate. In all, 100% DMSO was used as the negative control, and 10 mM mefloquine was used as the positive control. A 1000-fold dilution (either 100 nL or 50 nL) of compound from the master plate were spotted onto a 96-well or 384-well assay plate, respectively using Mosquito^® nanoliter dispenser (Cambridge, UK). The plates were then sealed with a removable foil seal using PlateLoc Thermal Microplate Sealer (Agilent) until use.

293-HA cells were infected with the mutant virus libraries and wild-type controls at an MOI of 0.1. At 1 h post infection, virus supernatant was removed and replaced with 0.3% bovine serum albumin (BSA)/MEM infection media. At 4 h post infection, cells were treated with 0.25% trypsin-EDTA and resuspended in 0.3% BSA/MEM. FACS was carried out using a FACSAria Cell Sorter (BD Biosciences) equipped with the FACSDiva software suite (BD Biosciences). A baseline fluorescence level was determined by analysing 10,000 cells infected with parental TY93/H5N1 GFP-627E virus. The cell sorting gate was then set to capture cells infected with a mutant virus library that expressed GFP at higher intensity than that observed for TY93/H5N1 GFP-627E. The cell sorting gate was adjusted every hour by analysing 10,000 cells infected with TY93/H5N1 GFP-627E virus. Single cells with increased GFP expression levels were sorted into 96-well plates seeded with 1.5 × 10⁵ MDCK-HA cells per well and incubated at 37 °C. At 48 or 72 h post infection, GFP levels were measured on an Infinit MH1000 plate reader (Tecan). Supernatants from GFP-positive wells were transferred to another 96-well plate by using a MICROLAB STAR liquid handling workstation (Hamilton), aseptically sealed with a PlateLoc Thermal Microplate Sealer (Agilent) and stored at −80 °C.

Tissue genomic DNA extraction kit DP304 (TIANGEN), KAPA HyperPlus Kits (Roche), HyperCap Bead Kit (Roche), SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 (Agilent), PlateLoc Thermal Microplate Sealer (Agilent), Herculase II Fusion DNA Polymerase Kit (Agilent), Sequencing and Library Building Platform (IIIumina USA) were used.