Cfx connect real time pcr detection system

Elongation rate experiments were performed generally as described (Singh & Padgett, 2009; Saponaro et al, 2014) were grown for 72 h with 4‐HT in 6‐well plates before the addition of 2 μM Flavopiridol (Sigma, F3055) for 4 h to inhibit transcription and allow full run‐off of elongating Pol II molecules. After two consecutive PBS washes, transcription inhibition was released by the addition of fresh complete medium. Every 5 min, one well was washed with PBS and directly lysed in TRIzol reagent (Thermo Fisher, 15596026) at −80°C. Total RNA was isolated following the instructions for the TRIzol reagent. 500 ng of total RNA was used for cDNA synthesis with the Superscript II RT enzyme (Thermo Fisher, 18064014) and random hexamer primers (Roche, 11034731001) following the manufacturer's instructions. RT–qPCRs were performed in triplicates in 20 μl total volume in a Bio‐Rad CFX Connect real‐time PCR detection system with GoTaq qPCR master‐mix (Promega, A6001). We used the C_t threshold cycle determined by the CFX Manager software in accordance with the 2−ΔΔCt method to analyze RT–qPCR data. The ratio relative to the untreated control sample is plotted.

After 72 h of 4‐HT treatment, WT and Spt5^dep MEFs were harvested and counted. Drosophila S2 cells were spiked in at a ratio of 4:1 (4 × 10⁵ MEFs and 1 × 10⁵Drosophila S2 cells). The cells were mixed and stored in TRIzol reagent (Thermo Fisher, 15596026) at −80°C. After obtaining total RNA according to the manufacturer's protocol, we treated the sample with RNase‐free DNaseI (Qiagen, 79254) in 100 ml reaction volume off the column according to the manufacturer's protocol. Following DNA removal, total RNA was again isolated with TRIzol. Next, cDNA synthesis was performed with the Superscript II RT enzyme (Thermo Fisher, 18064014) on 500 ng of DNase‐free total RNA with random hexamer primers (Roche, 11034731001) following manufacturer's instructions. RT–qPCRs were performed in triplicates in 20 μl total volume in a Bio‐Rad CFX Connect real‐time PCR detection system with GoTaq qPCR master‐mix (Promega, A6001). As a negative control to check for genomic DNA contamination, we ran samples not treated with Superscript II RT. We used the C_t threshold cycle determined by the CFX Manager software in accordance with the 2−ΔΔCt method to analyze RT–qPCR data, with Drosophila S2 act5c gene as spike‐in normalization reference.