Cellsdirect qrt pcr buffer

DSG-specific single B cells were sorted by FACS ARIA III into 96-well plates containing 10 µL Platinum Taq polymerase and SuperScript III reverse transcriptase (Invitrogen), a mixture of Taqman primer-probes at 0.2×concentration specific for the transcripts of interest and CellsDirect qRT-PCR buffer (Invitrogen). Immediately following cell sorting, samples were centrifuged, incubated at 55°C for 10 minutes, and subjected to 20 cycles of Polymerase Chain Reaction (PCR) (50°C for 15 minutes then 95°C for 15 seconds for the reverse transcription, followed by 20 cycles of 95°C for 15 seconds and 60°C for 4 minutes for amplification). Subsequent pre-amplified single-cell cDNA was stored at −20°C until analysis.

DSG-positive single B cells were sorted using FACS ARIA III into 96-well plates containing 10μL Platinum Taq polymerase and SuperScript III reverse transcriptase (Invitrogen), a mixture of Taqman primer-probes at 0.2× concentration specific for the transcripts of interest (Table SI) and CellsDirect qRT-PCR buffer (Invitrogen). Immediately after cell sorting, samples were centrifuged, incubated at 55°C for 10 min, and subjected to 30 cycles of PCR (50°C 15 min then 95°C for 15 s for the reverse transcription, followed by 30 cycles of 95°C 15 s, and 60°C 4 min for amplification). Subsequent pre-amplified single-cell cDNA was stored at −20°C until analysis.