Quantitative PUL Expression Analysis

PUL expression analyses were performed as previously described (24 (link)). Briefly, cells were harvested by centrifugation of the aliquots at 13,000 rpm for 10 min; cells were then treated with RNAprotect (Qiagen) and stored at −80°C until further processing. Total RNA was extracted from cells using an RNeasy minikit (Qiagen) and treated with Turbo DNase I (Ambion). Reverse transcription was performed using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. cDNA quantification was performed with a Mastercycler EP Realplex system (Eppendorf) using SYBR Fast qPCR master mix (Kapa Biosystems, Inc., Wilmington, MA) for 40 cycles of 95°C for 3 s, 55°C for 20 s, and 72°C for 8 s. All transcript levels were normalized based on 16S rRNA abundance, and transcript levels at time zero were used as references. Primers used are given in Table S2; they targeted previously validated sentinel susC-like genes, as previously reported (21 (link)).