Cellsdirect 2x reaction mix

Equal volumes of each inventoried TaqMan Gene Expression Assay (20X, Applied Biosystem) were pooled and then diluted using TE buffer so that each assay was at a final concentration of 0.2X. These pooled assays were for use in the pre-amplification step. Individual cells were harvested directly into the 10 µL RT-PreAmp Master Mix (5.0 µL CellsDirect 2X Reaction Mix (CellsDirect qRT-PCR kit, Invitrogen); 2.5 µL 0.2X Assay Pool; 0.2 µL RT/Taq Enzyme (CellsDirect qRT-PCR kit, Invitrogen); 2.3 µL Rnase-free water. The harvested single cell samples were immediately frozen and stored at −80°C. Cell lysis and sequence-specific reverse transcription were performed at 50 °C for 20 min. The reverse transcriptase was inactivated by heating to 95 °C for 2 min. Subsequently, in the same tube, cDNA went through sequence-specific amplification by denaturing at 95 °C for 15 s, and annealing at 60 °C for 4 min for 18 cycles. The pre-amplified products were diluted 5-fold and then analyzed by TaqMan PCR. Real-time reactions were performed with Universal PCR Master Mix and inventoried TaqMan Gene Expression Assays (Applied Biosystems) in 48.48 Dynamic Arrays on a BioMark System (Fluidigm). Threshold cycle (Ct) values were calculated from the system’s software (BioMark Real-time PCR Analysis, Fluidigm).

Sytox Red (ThermoFisher Scientific, Waltham, MA, United States) was added to cell suspensions to identify dead cells. Cells were sorted with a FACSARIA II (BD Biosciences). Debris and non-cellular particles were removed. Cell doublets and multiplets were removed using two consecutive gating steps: forward-scatter height (FSC-H) vs. forward-scatter area (FSC-A) and side-scatter area (SSC-A) vs. side–scatter width (SSC-W). Dead and compromised cells were identified by Sytox Red uptake and eliminated based on SSC-A vs. Sytox Red sorting. mGFP + /tdTomato-negative cells were collected into individual wells of 96-well PCR plates (USA Scientific, Ocala, FL, United States) pre-filled with CellsDirect 2 X Reaction mix (Invitrogen) and 0.05 U of SUPERase-In RNase Inhibitor (Invitrogen, Carlsbad, CA, United States). Flow rates were held at 300 cells/s (“precision” set to “single cell”; nozzle; 100 μm). Plates containing cells were sealed and stored at -80°C until RNA isolation.

Quantitative real time polymerase chain reaction (PCR) analysis was performed using the TaqMan gene expression assays (Life Technologies). CellsDirect One-Step qRT-PCR kit (Invitrogen) was used for cDNA synthesis and pre-amplification of target genes. Cells were FACS-sorted directly into 10 μL (bulk; 50 cells) of lysis/pre-amplification buffer. 10 μL of this buffer consisted of: 2.5 μL Taqman assay mix containing all assays at 0.2x dilution, 5 μL CellsDirect 2x Reaction mix (Invitrogen), 1.2 μL CellsDirect RT/Taq mix (Invitrogen), 1.2 μL TE buffer and 0.1 μL SUPERase-In RNase Inhibitor (Ambion). Reverse transcription and target pre-amplification were performed as follows: 50 °C for 15 min; 95 °C for 2 min; 22 cycles of 95 °C for 15 s and 60 °C for 4 min. Pre-amplified product was diluted 1:5 in TE buffer and analyzed using the ABI 7500 Fast real-time PCR instrument with specific Taqman probe (listed in Supplementary Table 6) that targeting Ezh2 deleted exon. Data were normalized to Hprt.