Table 1 Modifications to standard patch-clamp procedure for Patch-seq
Modification | Standard patch clamp | Patch-seq [1] | Patch-seq [2] | Purpose |
|---|---|---|---|---|
RNase-free precautions | − | ✔ | ✔ | Prevent sample degradation by exogenous RNase |
Large patch pipettes | − | ✔ | ✔ | Easier to aspirate cell contents into pipette |
Small volume of internal solution | − | ✔ | ✔ | Prevent interference with downstream reactions and loss of sample RNA |
EGTA in internal solution | − | ✔ | ✔ | Scavenge free calcium to reduce activity of endogenous RNase |
Glycogen in internal solution | − | ✔ | − | RNA carrier |
RNase inhibitor in internal solution | − | ✔ | − | Reduce activity of endogenous RNase |
Depolarizing current steps | +⁄− | +⁄− | ✔ | May facilitate diffusion of biocytin into cell and RNA into pipette |
Sequencing method | − | Smart-seq2-based | STRT-based |