1200 autosampler

P22-Cas9 and WT P22 were injected using an Agilent 1200 autosampler with 100 mM phosphate, 50 mM sodium chloride, pH 7.0 buffer. The buffer was degassed using an inline degasser. Samples were run over a WTC-100S5G guard column (Wyatt Technology Corporation) and a WTC-100S5 SEC column designed specifically for MALS (Wyatt Technology Corporation). The eluant was monitored using an in-line UV-Vis detector on the Agilent system as well as a Dawn Heleos 8 MALS detector and an Optilab T-rex RI detector (Wyatt Technology Corporation). All data were analyzed using ASTRA software from Wyatt. Samples were stored in the autosampler at room temperature, and the sample chamber in the RI detector was held at 25°C to reduce thermal drift. Molecular weights were determined from MALS and RI signals using the ASTRA software and dn/dc values of 0.185 was used for all proteins. The Cas9/P22 ratio was determined by subtracting the molecular weight of P22-Cas9 from P22 WT (see Figure 2c) and dividing by the molecular weight of Cas9.

Cells were stimulated with F. novicida or poly(dA:dT) for 5 h and snap-frozen. Cell extract prepared with methanol was filtered with 30 kDa filter to remove debris, dried, resuspended in water. c-di-AMP was added to the samples as an internal control. Samples were transferred to vials, capped and placed into the autosampler (kept at 4 °C). Aliquots of 10 or 20 µl from each vial was injected, along with cGAMP standards, onto the LC-MS/MS system (Agilent 1200 Autosampler and Binary pump 20) coupled to an ABI4000Q bench top mass spectrometer (MDS SCIEX). The MRM m/z transitions monitored for cGAMP and c-di-AMP were 675.5>506.0, and 659.0>524.0, respectively. Data were collected with the Analyst software.