Splicing assay of TREM2 minigenes

A splicing assay was performed as previously described^{42 (link),43 (link)}. Cells were cultured in 12-well plates and transfected with 0.5 μg of plasmids for U1/U7 snRNA expression (or cognate empty vector) and 0.05 μg of plasmid to express the minigene. Total RNA was purified using a NucleoSpin RNA kit (Takara Bio, Inc., Shiga, Japan) including DNase treatment. Total RNA (0.5 μg) was subjected to reverse-transcription using Revertra Ace -α- (Toyobo) with a 1:1 mixture of oligo dT and random hexamers as a primer. RT-PCR was performed using Blend Taq –plus- (Toyobo) and a panel of Fw and Rv primer sets as follows: EGFP-C1-Fw and TREM2-ex4-Rv for WT(ex2-4) and NHD(ex2-4) minigenes; TREM2-ex2-Fw2 and RT-TREM2-ex4-Rv for fl-WT and fl-NHD minigenes. PCR products were resolved by electrophoresis on 2% agarose gels or e-PAGEL polyacrylamide gels (ATTO, Tokyo, Japan) and the gels were stained with ethidium bromide (Genesee Scientific Corporation, San Diego, CA, USA) or GelRed (Wako). By sampling at multiple cycles, the cycle numbers of PCR were adjusted such that the amplification was within a logarithmic phase. The gel images were captured by either a digital camera (FAS-digi, Nippon Genetics, Tokyo, Japan) or CCD camera (WSE-6200H LuminoGraph II, ATTO). Multigauge software (FUJIFILM, Tokyo, Japan) was used to quantify the splicing patterns.