Investigation in the cannabigerol derivative VCE-003.2 as a disease- modifying agent in a mouse model of experimental synucleinopathy
Investigation in the cannabigerol derivative VCE-003.2 as a disease- modifying agent in a mouse model of experimental synucleinopathy
Animals were sacrificed by decapitation. Striatum was cold extracted and stored at -80C prior to RNA isolation.  RNA was isolated from the striatum samples using the RNeasy Lipid Tissue Mini kit (Qiagen, Barcelona, Spain) following the manufacturer's instructions, including the optional DNase I step. Once RNA was isolated, samples were stored at −80°C following library preparation. RNA concentration and RNA integrity number (RIN) was assessed using Nanodrop and Bioanalyzer (Centre for Genomic Regulation, Barcelona, Spain).  Libraries were prepared using the TruSeq stranded mRNA Library Prep (96 samples ref. 20020595 or 48 samples ref. 20020594) according to the manufacturer's protocol, to convert total RNA into a library of template molecules of known strand origin and suitable for subsequent cluster generation and DNA sequencing.  Briefly, 1000 or 500 ng of total RNA were used for poly(A)-mRNA selection using poly-T oligo attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented under elevated temperature and primed with random hexamers for cDNA synthesis. Then, the cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and random primers.  After that, second strand cDNA was synthesized, removing the RNA template and synthesizing a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA using DNA Polymerase I and RNase H. These cDNA fragments, then had the addition of a single 'A' base to the 3'ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation. A corresponding single T nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragments. Subsequent ligation of the multiple indexing adapter to the ends of the ds cDNA was done.  Finally, PCR selectively enriched those DNA fragments that had adapter molecules on both ends. The PCR was performed with a PCR Primer Cocktail that anneals to the ends of the adapters.  Final libraries were analyzed using Bioanalyzer DNA 1000 or Fragment Analyzer Standard Sensitivity (ref: 5067-1504 or ref: DNF-473, Agilent) to estimate the quantity and validate the size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit KK4835 (REF. 07960204001, Roche) prior to the amplification with Illumina's cBot Libraries were prepared using the TruSeq stranded mRNA Library Prep (96 samples ref. 20020595 or 48 samples ref. 20020594) according to the manufacturer's protocol, to convert total RNA into a library of template molecules of known strand origin and suitable for subsequent cluster generation and DNA sequencing.  Briefly, 1000 or 500 ng of total RNA were used for poly(A)-mRNA selection using poly-T oligo attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented under elevated temperature and primed with random hexamers for cDNA synthesis. Then, the cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and random primers.  After that, second strand cDNA was synthesized, removing the RNA template and synthesizing a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA using DNA Polymerase I and RNase H. These cDNA fragments, then had the addition of a single 'A' base to the 3'ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation. A corresponding single T nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragments. Subsequent ligation of the multiple indexing adapter to the ends of the ds cDNA was done.  Finally, PCR selectively enriched those DNA fragments that had adapter molecules on both ends. The PCR was performed with a PCR Primer Cocktail that anneals to the ends of the adapters.  Final libraries were analyzed using Bioanalyzer DNA 1000 or Fragment Analyzer Standard Sensitivity (ref: 5067-1504 or ref: DNF-473, Agilent) to estimate the quantity and validate the size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit KK4835 (REF. 07960204001, Roche) prior to the amplification with Illumina's cBot.