Smart-Seq2 Lysis Buffer
Required chemistry:
- Triton-X 100 (e.g. Sigma: PN 11332481001 or VWR: PN 1.12298.0101)
- Nuclease free water, not DEPC-treated! (e.g. UltraPure™ DNase/RNase-Free Distilled Water, Invitrogen: PN 10977035)
- (murine) RNase Inhibitor (NEB: PN M0314S)
Detergent dilution:
- dilute Triton-X 100 to a 0.2 % solution with nuclease free water (NFW):
- 2 µl Triton-X 100
- 998 µl NFW
- Lysis buffer master mix; scale up accordingly:
- 19 µl 0.2 % Triton-X 100 solution
- 1 µl RNase Inhibitor (40 U/µl)
The lysis buffer might be a bit foamy after mixing.
The lysis buffer has to be fresh
The lysis buffer has to be fresh at the moment of use, therefore prepare it shortly before collecting the cells. The buffer should be stored on ice until use or cooled during cell sorting workflows that take longer than 30 min. Freezing the lysis buffer at -20°C leads to inactivation of RNase Inhibitor and will reduce the quality of the RNA. The fresh solution is stable for at least 3h on ice.