5–4 Hz) EEG with no fluctuation in EMG. REM sleep was determined by low-amplitude and high-frequency EEG (similar to wake stage, but with rhythmic theta waves at 7–9 Hz) with low-amplitude

EMG. REM theta EEG power was analyzed with fast Fourier transform for band frequencies between 4–9 Hz. Sleep deprivation was initiated from ZT 0 for 6 hr with gentle handling. Mice were tested for spatial learning and memory using a Morris water maze as described with several modifications (Han et al., 2010). Mice were allowed to swim (1 min) during the training period (4 trials/day for 5 days) and then allowed to rest on the platform. During the examination day, mice were randomly placed in the three nontarget quadrants and allowed to swim for 1 min. For electrophysiology, hippocampal slices (∼400 μm) were processed and recordings obtained as described (Foster et al., 2008) (see Temozolomide order Supplemental Experimental Procedures). Mice (2–5 months) were also tested for seizure susceptibility after injection (40 mg/kg) with pentylenetetrazol. After injection, the mice were placed in an observational area for 60 min and the time of onset of convulsive behavior and nature and severity

of the convulsion were scored according to a modified Racine scale (Lüttjohann et al., 2009). For splicing microarrays and RNA-seq, hippocampal RNAs were obtained from Mbnl2+/+ and Mbnl2ΔE2/ΔE2 mice (2–3 months, n = 3 each). Splicing microarray analysis was performed as described ( Du et al., 2010) with modifications (see Supplemental Experimental Procedures). For RNA-seq, RNAs were purified and sequencing libraries were constructed using the www.selleckchem.com/Akt.html mRNA-Seq 8-Sample Prep Kit according to the manufacturer’s protocol (Illumina).

Libraries were sequenced (40 cycles, both ends) using an Illumina Genome Analyzer IIx. Raw sequence reads were mapped back to the mouse reference genome together with a database of annotated exon junctions compiled from mouse, human, and rat mRNA/EST data. CLIP was performed as reported ( Jensen and Darnell, 2008) with modifications (see Supplemental Experimental Procedures). Temporal cortex and cerebellar autopsy tissues (12 DM1 patients, 9 disease controls) were analyzed (Table S5). This research was approved by the Institutional Ethics Committee and written informed from consent for specimen research use was obtained from all patients. RNA was extracted using the ISOGEN procedure (Nippon Gene) and cDNA was synthesized using 1–3 μg of RNA. Random hexamers and cDNA equivalent to 20 ng RNA was PCR amplified for initial denaturation at 94°C for 10 min and 35 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s) (Table S6). PCR products were analyzed by capillary electrophoresis (Hitachi Electronics). The percentage of each peak was obtained by dividing each signal by the total signal and statistical analysis was performed using the Mann-Whitney U test.