Accuri cell analyzer

In-cell SHAPE-Seq measurements were carried out as described by Watters et al.^{35 (link)} Briefly, 3WJ repressor variants were transformed into BL21 Star DE3 as in the functional characterization experiments. Overnight cultures were diluted 100-fold into 1.2 mL of fresh LB with antibiotics. Following IPTG induction and 5 hr additional subculture, 100 µL of culture was removed and diluted by ~100-fold for functional characterization using a BD Accuri cell analyzer with a high-throughput sampler. 500 µL of the remaining culture was then added to 13.3 µL of 250 mM 1M7 or 13.3 µL of DMSO (control solvent). Cells were returned to shaking for 3 minutes to allow 1M7 to react, then cellular RNAs were Trizol extracted and reverse transcribed using a custom reverse transcription primer specific for GFPmut3b (5’-CAACAAGAATTGGGACAACTCCAGTG-3’). Additional 5’ and 3’ sequencing adapters were then added. Following 2 × 35 bp paired-end Illumina sequencing, ß reactivities were calculated as described by Aviran et al.^{50 (link)} Error bars represent the standard deviation of three samples, each probed from a separate transformation on a separate day. Replicate samples were only processed in parallel during final sequencing.