GSM4437142: D2 Con 1 input; Mus musculus; RNA-Seq
Six NAc tissue punches (2 mm) from two RT; D1-Cre or RT; D2-Cre mice are homogenized in 1 ml of homogenization buffer (Sanz et al., 2009) for each sample (2 mice, pooled for each sample). Samples are then centrifuged at 4°C, 10,000 g for 10 min and 750 μl of clear supernatants are mixed with 5 μg of HA-antibody (Abcam, #ab9110), and incubated on a gentle rotator at 4°C overnight. Remaining supernatants are saved for input samples at -80°C until RNA purification. The next day, 200 μl of magnetic beads (Dynabeads anti-rabbit IgG; Life technology) are added and incubated overnight with constant rotation. The following day, magnetic beads are washed 3 times in the magnetic rack for 10 min with a high salt wash buffer. Each RNA sample was extracted using Direct-zol miniprep kit (Zymo Research) and subjected to RNA-seq. For quality control, RNA integrity (RIN) values were measured and samples with RIN < 7 were excluded. Purified RNA samples from RiboTag immunoprecipitation (500 ng of RNA for each) were prepped for libraries after poly-A selection (KAPA Stranded mRNA-Seq Kit, Roche) and the libraries were sequenced in NextSeq500 (Illumina).
GEO Accession
GSM4437142