GSM5494572: P1_chchlearHC_Atoh1_C_R_rep2; Mus musculus; OTHER
P1 cochleas were dissected from hybrid by generated from Atoh1-GFP/Atoh1-GFP male mouse crossed with Atoh1-GFP/+ female mouse. Intact cochleas was dissected and directly used for antibody binding without enzyme digestion and single cell purification. Eeperimental and conrtol groups differ only in antibody.  Rabbit anti-GFP (ab290) antibody was used in experimental groups and  rabbit anti-mouse IgG (ab46540) antibidy was used in control groups. Bio-Mag Plus Concanavalin A coated beads were equilibrated and wash by binding buffer (20 mM HEPES pH7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) three times and resuspended by binding buffer. Fresh cochleas were transformed to 45 μl wash buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM spermidine, 1×proteinase inhibitor cocktail). Add 5 μl resuspended Co-A beads to each sample and incubate at room temperature for 10 min to ensure all beads bind to cochlea tissues . Then samples were separated from the buffer through magnetic stand. Samples were incubated with antibodies in a 1:100 dilutioned in antibody incubation buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.02% digitonin, 2 mM EDTA, 1× protease-inhibitor cocktails) at 4 °C for 2 h. After washing unbound antibodies, samples were incubated with pA-MNase for an additional hour at 4 °C. Then the sample were wash with dig-wash buffer to remove unbound pA-MNase. The pA-MNase cleavage were activated by adding 180 μl 2 mM CaCl2 for 15 minutes and stopped by adding 20 μl DR-Stop buffer (1.7 M NaCl, 100 mM EDTA, 20 mM EGTA, 0.02% Digtonin, 0.25 mg/ml glycogen). The cleaved Soluble soluble target DNA was allowed to release to the supernatant by incubating at room temperature for 15 minutes. After Ca2+ activationThe DNA was then purified through from the supernatant  by phenol-chloroform extraction in the presence of with 40 ng carrier RNA followed by ethanol precipitation overnight at -20 °C. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645S) was used for library construction. The library was size selected by 0.65× and another 0.75× beads.