GSM5332172: Control-4; Mus musculus; Bisulfite-Seq
Mice were anaesthetized with 0.12 mL euthanasia solution i.p., immediately followed by cervical dislocation and decapitation. Unfixed PFC tissue was roughly harvested, snap frozen in liquid nitrogen, and stored at -70°C until further processing. Batches of tissue punches were processed for nuclear isolation as described previously (Grindberg et al., 2013). Briefly, individual tissue punches were incubated in a Tris-buffered magnesium and potassium salt solution containing protease inhibitor and dithiothreitol. Tissue was triturated using a 1 ml pipette and was then homogenized using a polytron homogenizer. Triton-X100 was then added and the tissue Dounce homogenized using two pestles of decreasing size. Homogenate was strained through a 70 µm filter and centrifuged at 1000 x g for 8 min at 4°C. Nuclei pellet was resuspended, layered on a 29% Iodixanol gradient and centrifuged at 10,300 x g for 20 min at 4°C. Supernatant was collected, nuclei were counted from an aliquot. Nuclei in supernatant were incubated for 1 h with DAPI and anti-Satb2 antibody (1:100, abcam ab34735) conjugated to R-Phycoerythrin (RPE) using the Lightening-Link® R-Phycoerythrin conjugating kit (703-0010, Innova Biosciences), performed as per manufacturer's instructions. Triple positive (GFP, RPE and DAPI) nuclei were FACS-sorted and were spun down at 500 x g for 10 min at 4°C, nuclei pellet was snap frozen in liquid nitrogen and stored at -70°C until further processing. Reduced Representation Bisulfite Sequencing libraries were prepared as described previously (Bock et al., 2010).