Echo liquid handler

The Lincode siRNA Library (GE Dharmacon, G-301000) is a collection of siRNA reagents targeting 2231 human lncRNAs (1860 unique human lncRNA genes and 371 lncRNA transcripts associated with protein-coding genes). The design of this library is based on RefSeq version 54 and the siRNAs are arrayed as SMARTpools. The library was purchased at 0.1 nmol in a 96-well format. The library was diluted to a 5 μM stock with 1× siRNA buffer (GE Dharmacon, B-002000-UB-100) and arrayed onto black 384-well PerkinElmer Cell Carrier plates (Perkin Elmer, 6007550) using the Echo Liquid Handler (Labcyte). The black 384-well PerkinElmer Cell Carrier plates were prepared in advance and stored at −80 °C. The final siRNA concentration per well was 20 nM. An siRNA targeting exon 1 of lncRNA GNG12-AS1 (Silencer select, Life Technologies, S59962)^{70 (link)} and SMARTpool siRNAs targeting protein-coding gene CKAP5/Ch-TOG (GE Dharmacon, L-006847-00) were also included on each plate. CKAP5/Ch-TOG was used as a positive control as its depletion leads to mitotic delay and increased mitotic index^{24 (link)}. ECT2 SMARTpool siRNAs (GE Dharmacon, L-006450-00-0005) were used as a positive control in the third validation screen as its depletion results in multinucleated cells^{26 (link)}.

WM266-4 melanoma cells were pre labeled with 2μM CellTracker Orange CMTMR dye (ThermoFischer) as per manufacturer’s protocol. RNAi screens were performed in 96 well plates to which 160 nL/well siRNA (20 μM) were plated using an Echo liquid handler (LabCyte). Prior to seeding cells, 20 μL of OptiMEM (Invitrogen) containing 160 nL/well Lipofectamine RNAiMAX (Invitrogen) was added using a Multidrop Combi Reagent Dispenser (ThermoFischer) and plates were incubated for 30 min at room temperature (RT). One day after transfection cells were seeded on top of 100 μL of 1.8 mg/mL rat-tail collagen in DMEM plus 10% heat inactivated FBS prepared as per manufacturer protocol. Collagen was pre-aliquoted into wells of a 96-well, glass-bottomed, collagen-coated plates (PerkinElmer). After 24h of incubation cells were fixed by adding 100 μL of pre-warmed 8% methanol free formaldehyde (ThermoFischer) containing 5 μg/mL Hoescht stain (Invitrogen) and incubated for 1h at RT. Cells were imaged in the OPERA QEHS (PerkinElmer), with a 20x air immersion objective. Imaging was performed at a single timepoint capturing 15 wells per field and 200-plane z-stacks. Maximum intensity projections were used for image analysis.

For the HTS assay, plates (10 µM compound in each well) were prepared with an Echo liquid handler (Labcyte) at the FIMM (Institute for Molecular Medicine Finland) High‐Throughput Biomedicine unit. CSR3 and annealed siRNA were diluted to 200 and 750 nM, respectively, in reaction buffer (50 mM Tris‐HCl, 125 mM NaCl, 25 mM MgCl₂, pH 8.0). CSR3 solutions were dispensed into the wells of plates (10 µl CSR3 per well). Plates were incubated at room temperature for 15 min with shaking (450 rpm). Subsequently, 10 µl substrate was dispensed per well. The final CSR3 and substrate concentrations were 100 and 375 nM, respectively. The dose–response assay with six compound concentrations (range, 1.25 nM to 50 µM) was carried out with the same conditions. Plates were sealed, centrifuged briefly, and then, immediately analysed with a PHERAstar FS (BMG Labtech) with a fluorescence‐intensity optic module (excitation at 485 ± 6 nm, detection at 520 ± 5 nm) for 12 cycles (c.17 min total) at 37 °C. All dispensing was done using the BioTek MultiFlo FX. All screening plates contained positive‐control wells (lacking CSR3) and negative‐control wells (25 nl dimethyl sulphoxide, vehicle), which were used as standards to calculate the PI of compounds.

Total RNA was isolated using a TRI reagent. A total of 500 ng of total RNA was reverse-transcribed using a random hexamer primer and the MMLV Reverse Transcriptase Kit or 1000 ng of total RNA was reverse-transcribed using an EvoScript Universal cDNA. Quantitative PCR was carried out using the SYBR Green reagent and a LightCycler 480 apparatus (Roche, Meylan, France) or using TaqMan Gene Expression Assays and a LightCycler 1536 Instrument (Roche, Basel, Switzerland). Subsequently, 384-well plates were pipetted using Liquid Handling Robot (EpMotion 5070, Eppendorf, Hamburg, Germany), and 1536-well plates were pipetted using Echo Liquid Handler (Labcyte, Dublin, Ireland). The amplification specificity of the SYBR Green reagent was evaluated by determining the product melting curve. Target genes were determined using primers, as shown in Table 2. The relative mRNA expression was normalized to the expression of ribosomal protein lateral stalk subunit P0 (RPLP0) as a housekeeping gene. The expressions of the target genes were calculated by the ΔΔCT method [55 (link)] as fold change in the treatment groups relative to the control. All treatments were measured four times.