GSM5917167: retina, P4, NRL, 4; Mus musculus; OTHER
Retinas were dissected at P2, P4, P10, and P28 and cells were dissociated using dissociation solution (30 U/mL papain, HBSS pH 7.4, 1 mg/mL glucose, 10 mM Hepes, 100 U/mL DNase I, 5 µg/mL superoxide dismutase, 5 µg/mL catalase, 10 µg/mL D-alpha-tocopherol acetate, 1 mg/mL glucoscysteine-HCl, 5 µg/mL superoxide dismutase, 50 µg/mL gentamycin). Cells were pelleted at 200g for 5 min, washed and re-suspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM Spermidine) with Complete Protease Inhibitor tablet (MilliporeSigma, Burlington, MA, USA). Concanavalin A–coated beads (Polysciences, Warrington, PA, USA) were incubated with the cell suspension containing 250,000 cells for 10 min, washed to remove unbound cells, and incubated with NRL antibody (1:200) (Cat#ab-137193, Abcam, Cambridge, UK) in Antibody Buffer (20 mM EDTA and 0.02% digitonin in Wash Buffer) for overnight. Next day beads were washed with Wash Buffer containing 0.02% digitonin and incubated with 700 ng/ml pA-MNase for 1 hr at 4 °C. Beads were washed, re-suspended in 100 ul of Wash Buffer containing 0.02% digitonin and incubated with 2 mM CaCl2 at 0 °C for 30 min. Cleavage reaction was quenched by adding 100 ul 2X Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 25 μl RNase A, 0.05 mg glycogen and 10 pg heterologous spike-in DNA). DNA fragments were released from insoluble nuclear chromatin by incubation at 37°C for 10 min and extracted by spin column (Qiagen, Hilden, Germany). Libraries were prepared with ThruPLEX DNA-seq kit (Takara Bio USA, Inc, Mountain View, USA).