1 genetic analyser

The γδ TCR repertoire with regard to the main δ families (Vδ1, Vδ2, and Vδ3) and γ families (Vγ9, Vγ10, and Vγ11) was assessed using the spectratyping method adapted from Rådestad et al. [33 (link)]. Briefly, DNA was extracted from expanded UCB cells, and amplifications of 12 δ chain subfamilies and 9 γ chain subfamilies were performed by multiplex PCR reaction using primers as described before [34 (link)] (Tables
S1–3). The PCR was performed using AmpliTaq Gold 360 Master Mix (Applied Biosystems), specific primers in a final concentration of 200 or 400 nanoMoles as indicated in Table
S1 and
S2, and 100 ng of DNA, with a thermal cycler PCR machine (PTC-200, MJ Research, Watertown, MA). The process included the following steps: initial denaturation at 95°C for 10 minutes, followed by 35 cycles each of 94°C for 30 sec, 60°C for 45 sec, 72°C for 60 sec, and a final elongation step at 72°C for 10 minutes; and capillary electrophoresis where each PCR product was mixed with formamid (FA, HiDi Formamide) and size standard (GeneScan 400HD Rox Size std, Applied Biosystems) in 96-well MicroAmp plates (Applied Biosystems). Samples were analyzed using 3130 × 1 Genetic Analyser (Applied Biosystems). The results were analyzed using the PeakScanner software (Applied Biosystems).

Spa typing was performed as described by Harmsen et al. 12 (link) for all MRSA isolates. DNA sequencing was performed using a 3130×1 genetic analyser (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's protocol. Isolates were assigned to particular Spa types using the Spa typing website (http://www.spaserver.ridom.de).