Misonix 4000

Gata6-inducible mES cells were seeded at 1 × 10⁴ cells per square centimeter and treated with 1 µg/mL doxycycline for 36 h prior to harvesting. Immunoprecipitation was performed on 1 × 10⁷ to 2 × 10⁷ cells as described (Vokes et al. 2007 (link)) for three biological replicates versus input samples. Sonication was performed using a Misonix 4000 (28 cycles of 15 sec on and 45 sec off at an intensity of 70%) with a microtip probe (Misonix). The antibodies used are listed in Supplemental Table S7. Libraries were prepared using the TruSeq ChIP sample preparation kit, and the resulting samples were sequenced using the Illumina Genome Analyzer II (Illumina). Data will be deposited into Gene Expression Omnibus and released immediately after publication (GSE69323). Computational analysis details are included in the Supplemental Material.

Normal brain homogenates (NBH) from either human PrP (M¹²⁹ allele, tg650 line) [24 (link)] or ovine PrP (VRQ allele, tg338 line) [22 (link)] transgenic mice that over express PrP^C, 6 and 8 times respectively, were used as substrates for PMCA. After collection, mouse brains were rinsed in cold PBS and immediately frozen on dry ice before long-term storage at -80°C. Brains were then homogenized at a 10% (w/v) concentration in a conversion buffer composed of 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail (Roche) in PBS (pH 7.2). Homogenates were clarified at 2000 x g for 20 seconds and frozen at -80°C in single-experiment aliquots.
Wires were inserted into 0.2 ml PCR-tubes and mixed with 90 μl of 7.5% NBH in conversion buffer for Surf-PMCA amplification (Misonix 4000, N.Y., USA). One PMCA round was composed of 80 cycles of 20 seconds of sonication at 220–240 W power followed by 29 min 40 seconds of incubation at 37°C. Ten μl of amplified product was mixed with 90 μl of fresh NBH in conversion buffer and subjected to an additional PMCA round of 80 cycles. Three or four rounds were performed depending on the prion strain.

Experiments using PMCA were performed as previously described [75 (link)]. Briefly, HY TME, DY TME or 139H-infected brains were homogenized to 10% w/v in DPBS (Mediatech, Herndon, VA) or enriched PrP^Sc was used as a PMCA seed. Uninfected brain and spleen tissues were homogenized to 10% w/v and 20% w/v in conversion buffer respectively, and used as a PMCA substrate. PMCA seeds were diluted in PMCA substrate at a 1:100 ratio e.g. 1 μl of seed was diluted into 99 μl of substrate. PMCA was performed with a Misonix 4000 sonicator (Farmingdale, NY). For PMCA with uninfected brain as substrate the sonicator output set to level 75 and an average power output of 160 watts during each sonication cycle. For PMCA with uninfected spleen as substrate, the sonicator output was set to level 87 with an average power output of 220 watts during each sonication cycle. One round of PMCA consist of 144 cycles; one cycle comprises of five-second sonication followed by a ten-minute incubation at 37°C. All PMCA sample groups had an n = 3 and all experiments were replicated a minimum of three times.