Sarkosyl

Nascent transcription was captured in biological replicates by global nuclear run-on sequencing (GRO-seq) (Core et al., 2008 ) and nascent transcription start sites by 5′GRO-seq (Lam et al., 2013 ). Nuclei were isolated from BMDMs using hypotonic lysis [10 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, 3 mM CaCl₂; 0.1% IGEPAL CA-630] and flash frozen in GRO-freezing buffer [50 mM Tris- HCl (pH 7.8), 5 mM MgCl₂, 40% Glycerol]. 3–5 × 10⁶ BMDM nuclei were run-on with BrUTP-labelled NTPs as described (Duttke et al., 2015 (link)) with 3x NRO buffer [15mM Tris-Cl (pH 8.0), 7.5 mM MgCl₂, 1.5 mM DTT, 450 mM KCl, 0.3 U/μL of SUPERase-In (Ambion), 1.5% Sarkosyl, 366 μM ATP, GTP (Thermo Fisher Scientific), Br-UTP (Sigma Aldrich) and 1.2 μM CTP (Thermo Fisher Scientific, to limit run-on length to ~40 nt)]. Reactions were stopped after five minutes by addition of 500 μL Trizol LS reagent (Invitrogen), vortexed for 5 minutes and RNA extracted and precipitated as described by the manufacturer.

Brain and spleen homogenates were prepared at 10% in phosphate
saline buffer (PBS, MP Biomedicals, cat. no. 1860454) with a protease inhibitor
cocktail (Roche Diagnostics, cat. no. 11 697 498 001). After a brief
centrifugation to eliminate debris (805g for 45s in a Beckman-Coulter Allegra
25R centrifuge), supernatants were used for Western blot (WB) analysis. To
concentrate PrP^Sc and remove tissue components that may interfere
with PMCA, 500 µL of sample were mixed with a sarkosyl (Fisher
Bioreagents, cat. no. BP234) solution prepared in PBS (final concentration,
10%) and centrifuged at 100,000g for 1 h at 4°C [25 (link)]. Supernatants were discarded and PBS
was added to the pellets (without resuspension) in order to dilute out traces of
detergent. Samples were centrifuged again at 100,000g for 30 min at 4°C.
Final pellets were resuspended in 100 µL of normal hamster brain
homogenate prepared at 10% in Conversion Buffer (PMCA substrate, 150 mM
NaCl and 1% Triton X-100 in PBS) [8 (link);26 (link)] and submitted to
PMCA.