Rt buffer prep

Single-cell samples were pressure ejected into a fresh RT buffer prep (Applied biosystems). Samples were sonicated in a total volume of 20 µl at 40°C for 10 min before addition of RT enzyme mix (Applied Biosystem). Tubes were incubated at 37°C for 60 min and then 95°C for 5 min. Two rounds of amplification (30 cycles each) were done for the detection of Chat transcripts. For the first round of amplification (reaction volume 25 µl) included 2× mastermix, sterile water, 0.2 mM of each primer, 1 ml of cDNA sample. For the second amplification, the reaction included 1 µl of the previous (first-round) PCR product, 2× mastermix, sterile water, and 0.2 mM of each primer. Whole brain cDNA was run in parallel with the single-cell samples. After amplification, the PCR products (159 bp) were analyzed on 3% gels.

Single cell samples were pressure ejected into a fresh RT buffer prep (Applied biosystems). Samples were sonicated in a total volume of 20 μL at 40°C for 10 min before addition of RT enzyme mix (Applied Biosystem). Tubes were incubated at 37°C for 60 minutes and then 95°C for 5 minutes. Two rounds of amplification (30 cycles each) were done for the detection of Chat transcripts. For the first round of amplification (reaction volume 25 μL) included 2X mastermix, sterile water, 0.2 mM of each primer, 1 mL of cDNA sample). For the second amplification, the reaction included 1 μL of the previous (first-round) PCR product, 2X mastermix, sterile water, and 0.2 mM of each primer. Whole brain cDNA was run in parallel with the single cell samples. After amplification, the PCR products (159 bp) were analyzed on 3% gels.