title: METH
organism: Mus musculus
description: Protocols: Mice were given once daily injections of saline for two consecutive days (day 1-2), after which they were divided into two groups randomly. Two groups of mice were then given once daily injections of methamphetamine (METH, 2mg/kg) or saline for five consecutive days (day 3-7) followed by two injection-free days (day 8-9). On day 10, mice were given a challenge injection of either 2 mg/kg dose of METH or saline. Horizontal locomotor activity was performed on all drug treatment days for 60 minutes before and after the injections. For all experiments, mice were sacrificed 24 h after the last injection. After mice were sacrificed, nucleus accumbens (NAc) were harvested and NAc lysate from eight mice of each group were pooled as one sample for total RNA isolation, following to the manufacturers instructions of TRIzol (Invitrogen, USA). The RNA quality was evaluated by Agilent 2100 BioAnalyzer (Agilent Technologies, USA), and all samples exhibited an RIN > 8. Total RNA (5ug) of each sample was fragmented into ~200 base pair (bp) using a Covaris-S2 system after removed rRNA without preselecting mRNA. The first cDNA strand was synthesized using random hexamers and second strand cDNA was synthesized where dUTP was used instead of dTTP by DNA polymerase I. Then the double stranded cDNA were end-repaired after purified by QiaQuick PCR kit, and adapters were ligated. Subsequently, the uridine-containing strand was destructed by Uracil-N-Glycosylase, which enabled the identification of transcript orientation. After that, for acquiring the sequencing library products, the single stranded adapted cDNA fragments of 200 bp were recovered and purified with agarose gel electrophoresis and then enriched by PCR for 12 cycles.