Ssofast evergreen supermix

For each ChIP library, 50 ml of Mabs-S-Δlsr2-Clsr2-FLAG was grown until an OD₆₀₀ of 0.5 and fixed in 1% formaldehyde (Euromedex) and lysed using a Precellys grinder (3 cycles: 6,700 rpm −3 × 20 s ON/60 s OFF, Bertin Technologies) and VK05 beads. The bacterial chromatin was sheared in 100–300 bp fragments using a Bioruptor Pico (Diagenode). Chromatin Immunoprecipitation was performed as described previously (Grainger et al., 2006 (link)) using IgG M2 anti-FLAG (Sigma, F1804) and Anti-GFP (G6539, Sigma) mouse monoclonal antibodies attached to proteins A/G coupled to magnetic beads (Thermo fisher). The immunoprecipitated DNA and 1% of the total input were reverse-crosslinked and eluted using the iPure v2 kit (Diagenode). The quantitative PCR tests were performed using the SsoFast evergreen supermix (Bio-Rad). Enrichment of Lsr2 binding at the GPL operon operator was calculated using the “Percent Input” method using the primers MAB_4100c_qPCR1 and MAB_4100c_qPCR3 (Supplementary Table S1).

Fungal cells were grown overnight in YPD, washed once with phosphate buffer (pH 7.0), and resuspended in phosphate buffer. Approximately 4.5 × 10⁶ cells were inoculated in 5 ml YNBNP medium (0.67% YNB, 0.2% glucose, 25 mM phosphate buffer, 5 mM GlcNAc) at pH 7.0 and incubated at 37°C for 1 h. RNA was extracted using a MasterPure yeast RNA purification kit (Epicentre). DNA contamination was removed by the use of a Turbo DNA-free kit (Invitrogen). cDNA was synthesized from 500 ng DNase-treated RNA using a RevertAid H Minus First Strand cDNA synthesis kit (Thermo Scientific), following the manufacturer’s instructions for the random hexamer primer (IDT) and GC rich template. qRT-PCR was performed on a CFX96 real-time system (Bio-Rad), using SsoFast Evergreen Supermix (Bio-Rad) with the indicated primers to detect ACT1 (forward primer, 5′-ACTACCATGTTCCCAGGTATTG-3′; reverse primer, 5′-CCACCAATCCAGACAGAGTATT-3′) and RAS1 (forward primer, 5′-TATCAAGATGGATTAGCATTCG-3′; reverse primer, 5′-ATATTGGTCTTGACCTTGTTG-3′). Thermocycler conditions were as follows: 95°C for 30 s followed by 40 cycles of 95°C for 5 s, 65°C for 3 s, and 95°C for 5 s. RAS1 transcripts were normalized to ACT1 transcripts.

Target cDNA numbers were quantified using real time PCR using Ssofast Evergreen Supermix (Bio-Rad, CA, USA), target specific primers (Tables 1 and 2) and Cycler programs on the CFX96 Real time detection system (Bio-Rad laboratories, CA, USA) were created according to the manufacturer’s recommendations for optimized cycling conditions for RT-PCR. This comprised of an enzyme activation step at 95°C for 30 s (hot start), followed by 40 cycles of denaturation at 95°C for 5 s, annealing at specific primer annealing temperature (55–60°C, Tables 1 and 2) for 10 seconds and a melting curve analysis of 65–95°C with a plate read every 0.5°C at the end of the 40 cycles. Standard curves (ranging from 10⁷ to 10¹ copies per μL) were generated from plasmids of known DNA concentration to allow absolute quantification of target gene transcription. Each reaction was carried out in triplicate and the reaction efficacy was determined for each gene using ten-fold dilutions (10⁷ molecules/μl to 10¹ molecules/μl). Nuclease free water was used in place of DNA in triplicate as the non-template control. Absolute gene transcription was quantified by averaging triplicate quantities of each sample for all test genes, following normalisation of the expression ratio of each using the geometric mean of three reference genes.

Culture growth and RNA extraction was performed as described above for the RNA-seq analyses. RNA was DNAse treated with the Turbo DNA-free Kit (Invitrogen, Waltham, MA, USA). cDNA was synthesized from 500 ng DNAse-treated RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s instructions for random hexamer primer (IDT, Coralville, IA, USA) and GC rich template. qRT-PCR was performed on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA), using SsoFast Evergreen Supermix (Bio-Rad, Hercules, CA, USA) with the primers listed in Table S4. Thermocycler conditions were as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, 65°C for 3 s, and 95°C for 5 s. Transcripts were normalized to ACT1 expression.

Total RNA was isolated with the RNeasy kit (Qiagen) and reverse-transcribed into cDNA using the Superscript Strand III cDNA SuperMix (Invitrogen). Real-time PCR was performed using SSO Fast Evergreen Supermix (Biorad) on an Applied Biosystems 7500 Fast Real-Time PCR System at Functional Genomics Facility at Stanford. Expression of the target genes were normalized to the housekeeping gene 18S. Mouse RAW 264.7 cells and human PBMC were used as a positive control for the all the gene expression studies.
The target gene-specific primers were obtained from Integrated DNA Technologies (IDT). The primer sequences used in this study are shown in Supplementary Table 2. All real-time RT-PCRs were performed in triplicates and the relative mRNA expression of each target gene was determined by using the formula 2^−ΔCT (C_T, cycle threshold) where ΔC_T = C_T (target gene) − C_T (18S). The comparative expression level of each target gene between different samples was 2^−ΔΔCT.