I'm hoping to get a cold digest protocol working in my laboratory to perform single-cell RNA-Seq on various mouse tissues, based on e.g. Potter et al. 2019 Methods Mol. Biol, digestion of mouse kidneys. My first trial of Subtilisin A from Bacillus licheniformis was somewhat promising, except that the DNAse included in the digest buffer was clearly not very active at 4 degrees, and I lost about half of the cells in a single massive clump. The other problem was that one of the key cell surface markers needed for FACS index sorting was apparently degraded by the enzyme.
The digest buffer comprised 10 mg/ml Subtilisin A, 125 U/ml DNAse I and 5 uM CaCl2. Tissue was digested by a combination of shaking and trituration (1 ml pipette tip) at 4 degrees for approx. 60 minutes.
Can anyone share their experiences of working with cold-active proteases? Are there enzymes other than Subtilisin A that work well at 4 degrees? How can DNAse activity at low temperatures be improved?
Thanks!