Steponeplus qpcr machine

Total DNA and RNA were extracted from samples using TRiZOL reagent (Sigma Aldrich, Burlington, MA, USA) according to the manufacturer’s protocols. Synthesis of complementary DNA (cDNA) was carried out using MMuLV Reverse Transcriptase (NEB, Ipswich, MA, USA) and random hexamer primers (Integrated DNA Technologies, Coralville, IO, USA). Negative (no RT or no gDNA or cDNA synthesized from mock-infected cell supernatants) controls were used for each target per reaction. Quantitative PCR or RT-PCR analyses were performed using Brilliant III SYBR Green QPCR master mix (Bioline, Cincinnati, OH, USA) with gene-specific primers on an Applied Bioscience StepOnePlus qPCR machine (Life Technologies, Carlsbad, CA, USA). All primer sets were designed based on information present in existing literature [14 (link),17 (link),20 (link)]. Target gene expression levels were normalized to the endogenous 18S rRNA expression using the delta-delta comparative threshold method (ΔΔCT) (Table S1).

RT-qPCR of total RNA isolated from the 24-well format poly(I:C) transfections was performed using the Luna Universal One-Step RT-qPCR kit (New England BioLabs, Inc.) according to the manufacturer’s directions. Primer sequences can be found in Table 6. In brief, a master mix was prepared that comprised 10 μl of 2× Luna Universal One-Step Reaction Mix (2×), 1 μl of 20× Luna WarmStart RT Enzyme Mix, 0.8 μl of a 10 μM stock of each primer, and 5.4 μl of nuclease-free water per reaction. Each well received 18 μl of the appropriate master mix and 2 μl of the RNA being assayed. The following PCR program was then run on an Applied Biosystems Step One Plus qPCR machine (Life Technologies): denatured at 55°C for 10 min, 95°C for 1 min, followed by 40 cycles of 95°C for 10 s and 60°C for 60 s. Last, a melt curve was performed at 95°C for 10 s, 65°C for 10 s, 95°C for 10 s, and 50°C for 5 s.

Chromatin immunoprecipitation was performed as described (31 ). Briefly, yeast cultures were treated with formaldehyde (1% final) for 15 min before glycine (0.32% final) was added. The cells were washed twice in Tris-buffered saline (TBS) buffer, once in FA lysis buffer (pH 7.5, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate and 0.1% (w/v) sodium dodecylsulphate [SDS]), spun down and frozen at −80°C. Cell pellets were lysed in FA lysis buffer (with 2 mM phenylmethylsulfonyl fluoride, PMSF) by bead beating using a Precellys 24 homogenizer (Precellys). The cell lysates were incubated with anti-HA (ab9110, Abcam) or anti-Myc (ab32, Abcam) antibodies together with protein G Dynabeads (Invitrogen) on a wheel at 4°C overnight. The immunoprecipitates were washed five times in FA lysis buffer and eluted in ChIP elution buffer (50 mM Tris·Cl, pH 7.5, 10 mM EDTA and 1% (w/v) SDS). The samples were treated with proteinase K and incubated overnight at 62°C to reverse the crosslinks. Immunoprecipitated DNA was purified using PCR purification kit (QIAGEN) and quantified using the SYBR Green qPCR SuperMIX-UDG w/ROX kit (11744500, Invitrogen) by a SteponePlus qPCR machine (Life Technologies; all samples were also normalized by input DNA, quantified on the same plates).