GSM8244731: 16B_S7; Mus musculus; OTHER
Brains from male and female mice were extracted and flash frozen across 6 times of day (ZT2,6,10,14,18,22). Bilateral 1-mm punches were taken centered over the NAc. Co-immunoprecipitation, as described previously (37,38), was used to isolate the ribosome-associated mRNAs from D1- and D2-expressing neurons. NAc tissue was pooled from 2 Drd1- and A2a-Cre (D2) mice per sample. Tissue punches were dounce homogenized withbuffer containing 50 mmTris, pH 7.4, 100 mmKCl, 12 mmMgCl2, and 1% NP-40 supplemented with 1 mmDTT, 100 μg/ml cyclohexamide, 1 mg/ml heparin, RNasinâ Plus ribonuclease inhibitor (200 units/mL; Promega), and EDTA-free protein inhibitor cocktail (Roche). Samples were centrifuged at 10,000gfor 10 min and the clear supernatant was transferred to low-protein binding tubes. Anti-HA antibody (40mg; Biolegend) was added to each lysate sample. Tubes rotated overnight at 4°C. On day 2, Dynabeads (400 mL; Invitrogen) were washed in homogenization buffer (un-supplemented) and added to the antibody-bound lysate samples. Tubes were again incubated overnight at 4°C with rotation. On day 3, magnetic beads were washed 3 times in a magnetic rack for 5 min in high-salt buffer (50 mmTris, pH 7.4, 300 mmKCl, 12 mmMgCl2, 1% NP-40, 1 mmDTT, and 100 μg/ml cyclohexamide). Finally, RNA was extracted from the bead-bound polyribosomes by adding TRK lysis buffer provided in the MicroElute Total RNA Kit (Omega) according to the manufacturer's instructions. Eluted NAc cell-type specific RNA was used for RNA-sequencing. Astrocyte-specific mRNA was previously collected as above (38), except that 20 mg of anti-GFP antibody (Abcam) was used. Homogenate RNA was previously collected. Samples were collected across time of day and punched as above, but without pooling. Tissue was homogenized and total RNA was isolated using the RNeasy Plus Micro kit (QIAGEN). RNA quantity and quality for all samples were assessed using fluorometry (Qubit RNA High Sensitivity Assay kit; Invitrogen) and chromatography (2100 Bioanalyzer and RNA 6000 Pico kit; Agilent). Libraries were prepared with 10ng of RNA from each sample using the Smartseq HT ultra low input (D1 and D2) or Smartseq stranded total RNA ultra low input (homogenate) sample preparation kits (Illumina).