GSM5444756: P7_RodCells_H3K9me2; Mus musculus; OTHER
Retinal cells were isolated using papain isolation kit (Worthington Biochemical Corp, cat#LK003150). Isolation protocol was adopted from the manufacturer recommendations. Retina tissue was isolated and incubated with papain solution for 24-30 min at 37oC in with 600 rpm in a thermal shaker, then papain was blocked with a blocking solution. For CUT&RUN assay, live retina cells isolated from post-natal animals at day 7, 14, 21 and adult animals were flow sorted using Forward Scatter and Side Scatter. The CUT&RUN protocol was performed as previously described with modifications. Briefly, 1x106 cells (mESCs or rod cells isolated as described above) were harvested and washed twice with ice-cold PBS. Nuclei were isolated with Nuclear Extraction Buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol and 1x protease and phosphatase inhibitor). To aid in nuclear capture, isolated nuclei were incubated with activated Concanavalin A beads for 15 minutes at room temperature followed by incubation with the primary antibody or IgG control (1:100 dilution) overnight. Unbound antibody was removed with Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x protease phosphatase inhibitor) for a total of two washes. Supernatant was removed following bead-liquid separation using a magnetic stand and the fusion of proteinA-micrococcal nuclease (pA-MNase; Batch 6: Stock concentration: 143 μg/ml) was added at a final concentration of 700 ng/ml. The complex was incubated for 1 hour at 4°C. Following incubation, the bound complexes were washed twice with Wash Buffer and placed in a 0°C metal block for 5 minutes to pre-chill the tubes. To activate pA-MNase, CaCl2 was added to the pre-chilled bound complexes at a final concentration of 2 mM and incubated in the 0°C metal block for 30 minutes. The digestion reaction was neutralized by the addition of an equal volume of 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 40 μg/mL glycogen) followed by incubation at 37°C for 10 minutes to release cleaved DNA fragments. The targeted protein-DNA complex was released via centrifugation at 16,000 x g for 5 minutes and supernatant was collected and transferred to freshly labelled 1.7 mL lo-bind tubes. Digested DNA was purified via spin column (Macherey-Nagel, cat#740609.250) according to the manufacturer's instructions to recover large DNA-protein complexes. DNA concentration was estimated using the Qubit fluorometer. Protein A-MNase (Batch 6) was kindly provided by Dr. Steve Henikoff. The antibodies used were: Rabbit polyclonal anti-H3K9me2 (Active Motif, cat#39239); Rabbit Polyclonal Anti-H3K9me3 (Abcam, cat#ab8898); Rabbit polyclonal anti-H3K9ac (Active Motif, #39137), normal rabbit IgG (Cell Signaling, cat#2729s).