GSM5502166: 24PostSenPV_IP_AMPH_Isolation2_Sample2; Mus musculus; RNA-Seq
[Total RNA]  For each experiment, the two NAc samples from each single mouse were thawed in ice-cold homogenization buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH). The tissue was minced with a razor blade and dounce homogenized using a loose pestle in 1.5mL of homogenization buffer supplemented with 1mM DTT, 0.15mM spermine, 0.5mM spermidine, 172g/L kynurenic acid, and EDTA-free protease inhibitor. A 5% IGEPAL-630 solution was added to bring the homogenate to 0.3% IGEPAL-630, and the homogenate was further dounced with eight strokes of the tight pestle. When purifying RNA, RNaseOUT was added to all buffers at 60U/mL. When isolating nuclei for ATAC-seq, sodium butyrate was added to all buffers at a final concentration of 5mM. The sample was filtered through a 40μm strainer, mixed with 1.5mL of Working Solution (1:5 150mM KCl, 30mM MgCl2, 120mM Tricine-KOH, pH 7.8 Diluent and Optiprep Density Gradient Medium), underlaid with a gradient of 30% and 40% iodixanol, and centrifuged at 10,000xg for 18min on an Sw41Ti rotor in a swinging bucket centrifuge at 4°C. Nuclei were collected at the 30%-40% interface and pre-cleared by incubating with 20μL of protein G magnetic Dynabeads for 10min. After removing the beads with a magnet, the mixture was diluted with wash buffer (homogenization buffer plus 0.4% IGEPAL-630) and incubated with 10μL of 0.2mg/mL rabbit monoclonal anti-GFP antibody (Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526) for 30min. 60μL of Dynabeads were added, and the mixture was incubated for an additional 25 minutes. To increase yield, the bead-nuclei mixture was placed on a magnet for 30sec to 1min, completely resuspended by inversion, and placed back on the magnet. This was repeated 5-7 times. Samples were then re-placed on the magnet for 5min. 1mL of supernatant was removed as the Unbound Nuclear Fraction (UF) and placed on ice. The remaining beads were washed 3 times in 1mL of wash buffer followed by one wash in 6mL wash buffer. The 6mL was then sequentially applied to the magnet until all beads had been isolated. The final bead mixture was resuspended in 100µL wash buffer for downstream applications. All steps were performed on ice or in the cold room, and all incubations were carried out using an end-to-end rotator. 10µL of suspended UF nuclei were counted on a hemocytometer and 5000 UF nuclei were removed for use in downstream applications as an approximate reference sample to the number of PV+ nuclei harvested per mouse (calculated range 3000-5000/mouse). RNA isolation was performed using the RNaqeuous Micro kit (Thermo Fisher) according to the manufacturer's instructions, excepting that DNase digestion was not performed at this step, as the downstream library preparation included a DNase step. RNA was eluted in 15µL elution buffer from which 4µL was used for qRT-PCR gene enrichment validation. [RiboTag/Cytoplasmic RNA]  NAc tissue was rapidly dissected and Dounce homogenized in a tissue weight to buffer volume ratio of 5% (0.6-1mL) of homogenization buffer (HB-S, 50mM Tris, 100mM KCl, 12mM MgCl2, 1% NP-40) supplemented with 200U/mL RNAaseOUT, 1X Protease inhibitor, 1mM DTT, 100ug/mL Cyclohexamide, and 1mg/mL Heparin. Homogenate was spun down at 10,000 RPM and the supernatant was moved to the immunoprecipitation step after removal of 80µL for input vs IP comparisons. To prepare the antibody-bead mixture, Dynabeads were first washed in 1X PBS before 10µL Rabbit α-HA tag antibody (Abcam, #9110, RRID:AB_307019) was added to 300µL PBS+60µL Dynabeads and rotated at 4ºC for 4hr.  For immunoprecipitation, the remaining supernatant was subsequently combined with α-HA antibody-bead mixture and rotated overnight at 4ºC. Beads were isolated, washed 3 times in high-salt buffer, and lysed in Qiagen lysis buffer RLT for elution. RNA was subsequently extracted using the Qiagen RNeasy Mini Kit.  SMART-Seq™ v4 Ultra™ Low Input RNA Kit (Takara Bio #634888) was used to convert RNA to cDNA, which was then amplified and sequenced using an Illumina Hiseq 2500. [Total RNA]  Nugen Ovation Solo [RiboTag/Cytoplasmic RNA]  SMART-Seq™ v4 Ultra™ Low Input RNA Kit Total RNA-seq Ribo-Tag