PCR assays were performed using a custom panel of lymphoid development factors. Preamplified cDNA was obtained after reverse transcription (15’ at 40°C, 15’ at 50°C and 15’ at 60°C), and preamplification (22 cycles : 15’’ at 95°C, 4’ at 60°C), and diluted 1 : 5 in TE pH8 Buffer (Ambion). Sample mix was as follows : diluted cDNA (2.9 μl), Sample Loading Reagent (0.29 μl, Fluidigm), TaqmanUniversal PCR Master Mix (3.3μl, Applied Biosystem) or Solaris qPCR Low ROX Master Mix (3.3 μl, GE Dharmacon). Assay mix was as follows: Assay Loading Reagent (2.5μl, Fluidigm), Taqman (2.5μl, Applied Biosystem) or Solaris (2.5μl, GE Dharmacon). 48.48 or 96.96 dynamic array integrated fluidic circuit (IFC, Fluidigm) was primed with control line fluid, and the chip was loaded with assays (either Taqman or Solaris) and samples using an HX IFC controller (Fluidigm). The experiments were run on a BiomarkHD (Fluidigm) for amplification and detection (2’ at 50°C, 10’ for Taqman reagents or 15’ for Solaris reagents at 95°C, 40 cycles : 15’’ at 95°C, 60’’ at 60°C).
Hybridization protocol
n/a
Scan protocol
n/a
Description
Lin- IL7Rα+α4β7+PLZF-CXCR5-
Data processing
Normalization was performed using the average of three housekeeping genes: Actb, Gapdh, Hprt. Cells not expressing detectable levels of all three housekeeping genes were removed from further downstream analysis, along with a group of mast cell precursor contaminants. For quantification purposes, genes that were not detected were given a –ΔCt value of -30, close to the minimum value detected.
