NextSeq 500 sequencing; Effects of innate immune stimuli on PAF1 knockout cells
Effects of innate immune stimuli on PAF1 knockout cells
After 3 hours post-stimulation, all samples were collected with Trizol reagent (Ambion) and stored at -80ºC.  NcgRNA and PAF1 KO cells were plated at a density of 6.0 x 105 cells per well in 6-well plates and incubated at 37ºC for 24 hours. Cells were then stimulated with poly I:C (2.0 μg/mL, Tocris), poly dA:dT (1.0 μg/mL, InvivoGen), LPS (1.0 μg/mL, eBioscience), IFN-β (50 U/mL, R&D Systems), and TNF-α (100 ng/mL, Gibco Thermofisher) at the given concentrations. For poly I:C and poly dA:dT, transfection was performed with Lipofectamine 2000 (ThermoFisher) per manufacturer instruction. Total cellular RNA was extracted and purified with the RNeasy mini kit (Qiagen) At the sequencing facility, RNA samples were prepared with the QuantSeq3' mRNA-Seq Library kit for Illumina (Lexogen GmbH), following a standard protocol (Moll et al., 2014)