Epoch take 3 system

To determine decay of endogenous IFN-λ2/3 mRNAs, OGG1 proficient, OGG1 KO cells or hSEACs with functionally (TH5487) inactivated OGG1 were poly(I:C) treated for 30 min and monolayers were washed with PBS. Four hours later 20 µg/mL 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB; D1916; Millipore-Sigma, Saint Louis, Missouri, USA) was added. Total RNAs were isolated at DRB addition (0 h), 2, 4, 8 and 18 h using a RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Total crude RNAs were DNaseI-treated and loaded onto a RNeasy column and subjected to washes. RNAs eluted with the RNase-free water included in the kit. The RNA concentration was determined spectrophotometrically on an Epoch Take-3™ system (Biotek, Winooski, VT) using Gen5 v2.01 software. The quality of the total RNA was confirmed via the 260/280 nm ratio, which varied from 1.9 – 2.0. 0.5 µg RNA was used to generate cDNA with oligo-dT (Takara, RR037A). The quantities IFNL2/3 mRNAs at each time points were determined by qPCR by normalizing to 18S rRNA. The relative amount of IFN-λ2/3 mRNA at time 0 h of DRB addition was set at 100% in each cell type (OGG1 proficient, OGG1 KO cells or hSEACs with functionally (TH5487) inactivated OGG1).

Total RNAs were extracted using a RNeasy Mini kit (Qiagen, 74106) according to the manufacturer’s instructions. Crude RNAs were DNaseI-treated and loaded onto a RNeasy column and subjected to washes with RW1 and RPE buffers. The RNA concentration was determined spectrophotometrically on an Epoch Take-3™ system (Biotek, Winooski, VT) using Gen5 v2.01 software. The quality of the total RNA was confirmed via the 260/280 nm ratio, which varied from 1.9 to 2.0. 500 ng total RNA was used to generate cDNA using iScript reverse transcription supermix (Bio Rad, 1708840). qPCR was performed using specific primers, and cellular GAPDH as an internal control (sequences of primer are listed in Supplementary Table 1). Changes in mRNA levels were calculated using the 2-ΔΔCt method.