Viromer green

BMDMs were reverse transfected with siRNAs against screen hit genes using Viromer Green (Lipocalyx, cat# VG-01LB-00). 5 μl of siRNAs (0.5 μM) were spotted in 96-well plates (BD Falcon, cat# 353219), and 0.1 μl of Viromer Green transfection reagent pre-mixed with 4.9 μl of Viromer Green Buffer was added to each well. Plates were tapped to mix the transfection reagents and centrifuged at 400 g for 1 s. After incubation for 30 min at room temperature, 40,000 BMDMs in 90 μl of complete DMEM were seeded per well for a final siRNA concentration of 25 nM. Plates were incubated at room temperature for 10 min to allow the cells to settle, then at 37°C in a humidified atmosphere with 5% CO₂ for 48 hr. Cells were stimulated with 5 nM lipid A (Avanti Polar Lipids, Alabaster, AL, cat# 699500P) for time periods as indicated. Cells from 24 wells were pooled for total RNA extraction with RNeasy Mini Kit (Qiagen), then reverse transcribed with iScript cDNA synthesize kit (Biorad, cat# 1708891). For measurement of secreted protein level, supernatants were collected and subject to ELISA as described above. Selected siRNAs for the 4 hit genes were all from Thermofisher with the following siRNA IDs: s106096 and 298393 for Helz2; s104443 and s104445 for Phf11d, s100717 and 163480 for Sertad3, s76360 and s76361 for Zscan12.