Illumina HiSeq 4000 sequencing; RNA-sequencing of dorsomedial striaum in male Drd2 flox/flox (WT) and Drd2 flox/flox ChAT-Cre+/- (ChI-D2RKO) mice treated with saline or 20 mg/kg of cocaine (ip) 1 hour prior to dissection
RNA-sequencing of dorsomedial striaum in male Drd2 flox/flox (WT) and Drd2 flox/flox ChAT-Cre+/- (ChI-D2RKO) mice treated with saline or 20 mg/kg of cocaine (ip) 1 hour prior to dissection
Mice were habituated in a novel home cage for 2 hours. They were then injected with saline or cocaine (20 mg/kg; ip). 1 hour later, they were sacrificed via cervical dislocation and their brains were rapidly dissected. Once removed, the brains were rapidly frozen in 2-methylbutane. Tissue punches of the dorsomedial striatum were obtained for RNA isolation. After adding Trizol, the tissue was homogenized rapidly using a 26 ½ gauge needle attached to a 1 mL syringe. Chloroform was added and the samples were thoroughly mixed followed by centrifugation. 20 ug of glycogen, sodium acetate pH 5.2 (0.3M final concentration), and 2.5 volumes of isopropanol were then added to the aqueous phase. RNA was precipitated at room temperature for 10 minutes before centrifugation at 4C for 10 minutes. The resulting pellet was resuspended in water and sodium acetate pH 5.2 was added (0.3M final concentration). A 25:24:1 mix of phenol (pH 6.7), chloroform, and isoamyl alcohol was added; the samples were vigorously mixed and centrifuged briefly. A mixture of 24:1 chloroform and isoamyl alcohol was added to the resulting aqueous layer and was vortexed vigorously. 20 ng of glycogen, sodium acetate (0.3M final concentration), and 2.5 volumes of 100% ethanol were added the aqueous layer and were precipitated at -80C for 1 hour. The samples were centrifuged at 4C for 10 minutes and the resulting pellet was washed once with 75% ethanol. The pellet was resuspended and water and quantified on a nanodrop. Total RNA was monitored for quality control using the Nanodrop absorbance ratios for 260/280nm and 260/230nm and the Agilent Bioanalyzer Nano RNA chip (Table 1). Library construction was performed according to the Illumina TruSeq® Stranded mRNA Sample Preparation Guide.  The input quantity for total RNA was 150ng and mRNA was enriched using oligo dT magnetic beads. The enriched mRNA was chemically fragmented for three minutes.  First strand synthesis used random primers and reverse transcriptase to make cDNA.  After second strand synthesis the double stranded cDNA was cleaned using AMPure XP beads and the cDNA was end repaired and then the 3' ends were adenylated.  Illumina barcoded adapters were ligated on the ends and the adapter ligated fragments were enriched by nine cycles of PCR.
Experimental Factor: compound
saline
Experimental Factor: dose
0
Experimental Factor: genotype
Drd2 flox/flox