GSM5233838: RARA_Input; Mus musculus; ChIP-Seq
6x10e5 low passage (< 10) E14 cells were plated per 15 cm culture dish and cultivated for 48 h in regular ES medium containing LIF. 4 h prior to cross-link cells were treated with RA as described in the treatment protocol. Then cells were washed with PBS, trypsinized and gently but thoroughly resuspended in ES medium to obtain a single cell suspension of  12x106 cells in 10 ml medium. Cells were cross-linked adding 1% formaldehyde for 5 min and the reaction was quenched using 125 mM glycine for another 5 min. After two wash steps with PBS, nuclei were prepared by washing twice with ice-cold Lysis Buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1x proteinase inhibitor cocktail (PIC, Roche)), and finally cell pellets were resuspended in 100 µl Sonication Buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1x PIC) per 2x106 cells, incubated for 20 min on ice, homogenized 10 times with a 27G needle, and chromatin was sheared by sonication applying a Bioruptor Pico (Diagenode) device for 40 cycles (30 sec pulse, 30 sec pause). Cell debris was removed by centrifugation at maximum speed for 10 min at 4°C and chromatin containing supernatants were used for IP. To prepare input DNA, 30 μl aliquots (1/10 of the amount used per IP) were saved at -20°C. For IPs 300 μl chromatin (equals 6x 106 cells) were incubated with 4 μl of RAR antibody (Diagenode C15310155, polyclonal rabbit IgG, Lot # A704-001) at 4°C overnight under rotation. Protein-A/-G agarose (sc-2003) was equilibrated with sonication buffer, 30 µl 1:1 slurry was added to each sample and incubated at 4°C for 4 h under constant rotation. Beads were extensively washed with: 1x Wash Buffer I (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, pH 8.0, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1x PIC), 1x Wash Buffer II (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, pH 8.0, 1% Triton X-100, 500 mM NaCl, 0.1% SDS, 1x PIC), 1x Wash Buffer III (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1x PIC) for 5 min under rotation, and 2x with TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) for 1 min. Protein-DNA complexes were eluted with 100 μl Elution Buffer (25 mM Tris-HCl, pH 7.5, 10 mM EDTA, pH 8.0, 1% SDS, 100 mM NaCl). Input samples were adjusted to 100 μl with Elution Buffer. Eluates and input samples were first incubated with RNase A (100 µg/ml final) for 1 h at 37°C and then with Proteinase K (200 µg/ml final, Ambion) for 1 h at 50°C. Cross-linking was reversed by incubation at 65°C overnight. DNA was recovered using Zymo ChIP DNA clean & concentrator kit (D5202). Sequencing libraries for RAR ChIP and Input fragments were prepared using the KAPA Hyper Prep Kit (Roche).