REPLI-g Advanced DNA Single Cell Kit (96)

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Protocol tips
Follow manufacturer’s instructions

Publication protocol

Generation of amplicons: GFP-positive nuclei were sorted using a BD AriaFACS III into a PCR tube strip containing REPLI-g Advanced Single Cell Storage buffer (QIAGEN). Whole genome amplification (WGA) was performed on the samples directly following FACS using the REPLI-g Advanced DNA Single Cell kit (QIAGEN) according to manufacturer’s instructions.
For generation of the specific amplicons, 1ul of WGA DNA was diluted 1:50 and amplified (PCR 1) with Phusion High Fidelity Polymerase (Thermo Fisher) using the following protocol (all temperatures in Celsius). Initial denaturation at 95 degrees for 30 s fol- lowed by 34 cycles of denaturation at 95 degrees for 10 s, annealing at 66 degrees for 20 s, and extension at 73 degrees for 10 s. A final extension was performed at 72 degrees for 5 min.
The product of PCR 1 was diluted 1:100 and amplified again (PCR 2) with a second set of primers to decrease non-specific amplification and increase amplified DNA yield using the thermocycler protocol. The amplicons were gel extracted using the MinElute gel extraction kit (QIAGEN), and stored at 20C or directly used in downstream processing.

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