title: GSM5332173: PFC DKO-1; Mus musculus; Bisulfite-Seq
library_construction_protocol: 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).
attributes.tag: GEO Accession
attributes.value: GSM5332173