Neb protoscript 2 first strand cdna synthesis kit

The calibration of reporter expression by RT-qPCR shown in Supplementary Figure 2 was performed as described in (1) . In brief, cells were grown to OD 450 0.2 in M9RDM, at which point 650 μl of the culture was mixed with 1.3 mL RNAProtect Bacterial Reagent (Qiagen) and frozen according to the manufacturer's instructions. RNA was then purified using the RNeasy mini kit (Qiagen). An additional DNaseI treatment (Qiagen RNase-Free DNase Set) was performed on the purified sample in order to remove residual gDNA and re-purified using the RNeasy kit. cDNA was finally synthesized using the NEB Protoscript II First Strand cDNA synthesis kit (New England Biolabs) following the manufacturer's instructions in parallel to control samples without the Reverse Transcriptase enzyme (-RT). qPCR reactions on the cDNA and -RT control samples described above were performed with primers for mNG 63-64 and mdoG primers 65-66 (see Supplementary Data 3 ) using BioRad iTaq Universal SYBR Green Supermix, on a BioRad CFX96 instrument. In all cases the -RT samples were at least 6 Ct higher than the matched cDNA sample (comparable to water-only controls). In each case, we calculated the expression levels based on the Ct values obtained for the mNG reporter in the cDNA sample normalized by the signal from mdoG.