Single-cell RT-PCR(nest PCR)

Following the reverse transcription reaction (20 µL), 10 µL was used for the first PCR, and the remaining 10 µL was stored at −80 °C as a backup.

1st PCR

Primer mix

Reaction

1. 94℃ 2min;
2. 95℃ 30s;
3. 65℃ 10min
4. Go to 2, 19cycles;
5. 72℃ 10min

2nd PCR

There is no PCR clean between 2 steps

Reaction(For example, pcdhga1, different isoform need different primer F)

1. 95℃ 5min;
2. 95℃ 30s;
3. 62℃ 30s;
4. 72℃ 1min;
5. Go to 2, 34 cycles;
6. 72℃ 10min

An aliquot of 8.5 µL was taken for agarose gel electrophoresis.