Sample 8
Protocols: samples were collected directly in TRIzol™ Reagent (Invitrogen, 15596-018) 350-μm thick cerebellar slices were produced using a Leica vibratome and temporarily kept in ice-cold Gey's balanced salt solution (GBSS) supplemented with kynurenic acid (1 mM) and glucose. Slices with intact morphology were then collected and exposed to RML6 prion or NBH brain homogenate for 1 hour at 4 °C. After extensive washes, six to eight slices were put on a Millicell-CM Biopore PTFE membrane insert (Millipore) and kept on slice culture medium (50% vol/vol MEM, 25% vol/vol basal medium Eagle and 25% vol/vol horse serum supplemented with 0.65% glucose (w/vol), penicillin/streptomycin and glutamax (Invitrogen)) at 37 °C in a cell culture incubator. Culture medium was changed three times per week. Anti-PrP antibody FabPOM2 was supplemented in the slice culture medium with 500 nM concentration from the second day after the cultures were established. The treatment continued until to the end of the experiment. Fresh FabPOM2 was added in the culture medium every time when the culture medium was changed. Total RNA was extracted according to the instruction of TRIzol™ Reagent (Invitrogen, 15596-018). After cleanup with the RNeasy Plus universal mini kit (QIAGEN) and quality-check with Bioanalyzer 2100 (Agilent Technologies), sample mRNA passed the quality control was enriched with the poly-A method. Sequencing libraries were then prepared with the TruSeq RNA Sample Prep kit v2 (Illumina)