How do I lyse cells for direct cDNA synthesis?
How do I lyse cells for direct cDNA synthesis?
Refer to the user manual for the specific kit that you are using, as the lysis reaction conditions may be different for different kits. For the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing or the SMARTer Ultra Low Input RNA Kit for Sequencing - v3, lysis is conducted at room temperature, while for the previous generations it is done on ice. Lyse the collected cell(s) with Reaction Buffer (Dilution Buffer + RNase Inhibitor) and incubate at room temperature or on ice for 5 minutes.Since the Reaction Buffer contains RNase Inhibitor, we strongly recommend preparing it immediately before use. If it is not feasible to prepare the Reaction Buffer immediately before use, you may keep it on ice and add RNase Inhibitor immediately before use.
Note: Dilution Buffer contains a detergent; therefore, mix it carefully to avoid bubbles.