RNA_Seq of Mus musculus: DAT:TH KO adult male ventral tegmental area
Mice with inactivation of the Th gene in DAT-expressing neurons (DAT:TH KO) were generated by crossing mice with two floxed Th alleles (Thlox/lox) with mice that have one deleted Th allele (Th /+) and one allele with Cre recombinase expressed under the control of the Slc6a3 gene encoding DAT (Slc6a3Cre/+). DAT:TH KO mice had the genotype Th /lox-Slc6a3Cre/+. Mice, 3-5-month-old, were sacrificed by decapitation after brief exposure to CO2; the VS, DS, SNc, VTA and PFC from each mouse were dissected from the brain. Tissues were immediately frozen in liquid nitrogen and stored at 80 C. Tissues were processed, and subsequently cDNA libraries from isolated RNA. Each time tissues were homogenized using Bullet Blender with 0.5 mm RNase Free Zirconium Oxide Beads (Next Advance) in RLT buffer from RNeasy Plus Micro Kit (Qiagen). Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen). The quality and quantity of RNA from each sample was determined using an Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Chips. Messenger RNA was then obtained from at least 100 ng of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina following manufacturers protocol. The library quality and insert length were checked using the Agilent 2100 Bioanalyzer with DNA High Sensitivity DNA Kit. The libraries were sequenced using Illumina HiSeq 2000. The quality of obtained fastq files was examined using FASTQC. The reads were aligned to the mouse reference genome (GRCm38) using HISAT2 v2.1.0 aligner with --rna-strandness RF parameter.