Lightcycler 480 instrument

For expression analyses of tissue samples, mice were killed by cervical dislocation. Samples were quickly cooled and region of interest prepared. RNA was extracted using RNeasy Mini (Qiagen). cDNA was synthesized with Superscript III (Invitrogen). Concentration and quality of RNA was evaluated using a NanoDrop spectrophotometer and RNA Nano (Agilent). RNA from MACS-purified cells was extracted using QIAshredder and RNeasy protocols (Qiagen). cDNA was amplified by Single Primer Isothermal Amplification (Ribo-SPIA® technology) using Ovation PicoSL WTA System V2 (NuGEN) following the manufactures protocol. Quantitative PCRs were done in triplicates on 384-well plates using the GoTaq® qPCR Master Mix (Promega, A6002) and the LightCycler® 480 Instrument. Background subtraction and thresholding was performed using the LightCycler® 480 software 1.5 (Roche)^{56 (link)}. Expression values were normalized to the mean of at least 2 out of the housekeeping genes Hprt, Rps13, Rplp0, Gapdh, 18S (Extended Data Fig. 2a, 5e, 9o, 10a). Quantification was done by applying the ΔΔCt method, normalized to experimental controls (set to 1). All primers (Supplementary Table 4) were designed to fulfill optimal criteria e.g. primer length (18-22 bp), melting temperature (52-58°C), GC content (40-60%), low number of repeats, amplicon length (<220 bp). All primers were intron-spanning.