TREM2 protein, human

Details of PSC lines used in this study can be found in Supplemental Experimental Procedures. TREM2 mutant lines were generated from fibroblasts derived from skin punch biopsies obtained following informed consent, after approval from the ethics committee of the Istanbul Faculty of Medicine, Istanbul University, or the joint research ethics committee of the National Hospital for Neurology and Neurosurgery and the Institute of Neurology, UCL. Microglia were differentiated from PSCs via embryoid bodies and PMPs (Karlsson et al., 2008 (link), van Wilgenburg et al., 2013 (link)). In brief, at least 2 days after last passaging, PSCs (cultured feeder-free in E8) were passaged to single cells with TrypLE Express (Gibco), and plated at 10,000 per well in 96-well ultra-low attachment plates (Corning) in 100 μL embryoid body medium (10 μM ROCK inhibitor, 50 ng/mL BMP-4, 20 ng/mL SCF, and 50 ng/mL VEGF-121 in E8), before centrifugation at 300 × g for 3 min. Embryoid bodies were cultured for 4 days, with half medium change after 2 days. Ten to 16 embryoid bodies were plated per well of tissue culture-treated 6-well plates and cultured in 3 mL hematopoetic medium (2 mM GlutaMax, 100 U/mL penicillin, 100 μg/mL streptomycin, 55 μM β-mercaptoethanol, 100 ng/mL M-CSF, and 25 ng/mL IL-3 in X-VIVO 15 [Lonza, LZBE02-060F]). From this point on, 2 mL medium was exchanged every 4–7 days.
PMPs were harvested from suspension during medium exchange and plated in RPMI 1640 at 180,000 cells/cm² in 6-, 12-, or 96-well plates. In the absence of serum, PMPs adhered to uncoated tissue culture-treated plastic within 1 hr, at which point medium was switched to complete microglia medium (10% FBS [Gibco, 16000044 or Sigma, [F2442], 2 mM GlutaMax, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ng/mL IL-34, and 10 ng/mL GM-CSF in RPMI1640).
Final differentiation of PMPs into microglia occurred over 6–10 days, with full medium change every 2–3 days. All cytokines and growth factors obtained from PeproTech, except for IL-3 (Cell Guidance Systems).

Selfie RT-qPCR that measures the absolute number of RNA transcripts per gene was performed as described by Podlesniy and Trullas [33 (link)]. Briefly, cells were extracted and diluted in the 100ST buffer (DireCtQuant, Lleida, Spain, DCQ100ST) following the manufacturer’s instructions. Then, a pre-annealing step was performed per duplicate, by mixing 2 μl of the sample with the same volume of 2.5 μM of the corresponding reverse primer (Trem2 5′-ctcggagactctgacactgg-3′; Clec7a 5′-gcactgcagcaaccactact-3′) at 70 °C for 5 min. Next, the reaction mixture containing 1 mM dNTP mixture, 10 U of the RiboLock RNase Inhibitor (Thermo Fisher Sci., EO0381) and 200 U of Maxima H Minus Reverse Transcriptase (Thermo Fisher Sci., EP0751) or 50% glycerol was retro-transcribed for 30 min at 60 °C followed by 5 min at 85 °C. Finally, 2.5 μM of the corresponding forward primer (Trem2 5′-tggaaccgtcaccatcactc-3′; Clec7a 5′-cttcaccttggaggcccatt-3′) was added and amplified by conventional RT-qPCR (5 min at 95 °C, followed by 49 cycles of 15 s at 95 °C, 25 s at 60 °C and 25 s at 72 °C), using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725271). The number of transcripts per encoding gene was calculated as the fold change after subtracting the Ct values obtained from the sample containing glycerol.

Selfie RT-qPCR that measures the absolute number of RNA transcripts per gene was performed as described by Podlesniy and Trullas [33 (link)]. Briefly, cells were extracted and diluted in the 100ST buffer (DireCtQuant, Lleida, Spain, DCQ100ST) following the manufacturer’s instructions. Then, a pre-annealing step was performed per duplicate, by mixing 2 μl of the sample with the same volume of 2.5 μM of the corresponding reverse primer (Trem2 5′-ctcggagactctgacactgg-3′; Clec7a 5′-gcactgcagcaaccactact-3′) at 70 °C for 5 min. Next, the reaction mixture containing 1 mM dNTP mixture, 10 U of the RiboLock RNase Inhibitor (Thermo Fisher Sci., EO0381) and 200 U of Maxima H Minus Reverse Transcriptase (Thermo Fisher Sci., EP0751) or 50% glycerol was retro-transcribed for 30 min at 60 °C followed by 5 min at 85 °C. Finally, 2.5 μM of the corresponding forward primer (Trem2 5′-tggaaccgtcaccatcactc-3′; Clec7a 5′-cttcaccttggaggcccatt-3′) was added and amplified by conventional RT-qPCR (5 min at 95 °C, followed by 49 cycles of 15 s at 95 °C, 25 s at 60 °C and 25 s at 72 °C), using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725271). The number of transcripts per encoding gene was calculated as the fold change after subtracting the Ct values obtained from the sample containing glycerol.