Anti rabbit igg control antibodies

Mouse N2A neuroblastoma and E14 ES cells were cultured as described in (Vance et al., 2014 (link)). The N2A cell line was
chosen because it has been used extensively as a model to study neural
differentiation in vitro (Shea et al.,
1985). Human neuroblastoma (SH-SY5Y) cells were grown in DMEM/F12 medium
supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at
37°C in a humidified atmosphere with 5% CO₂. Biochemical
fractionation, ChIP and UV-RIP experiments was performed exactly as described in
Vance et al. (2014). (link) The following
antibodies were used: anti-DNMT1 (ab87656; Abcam, UK), anti-BRG1 (ab4081; Abcam),
anti-P66beta (ab76924; Abcam), anti-SIN3A (Active Motif, Belgium, 39,865), anti-CTCF
(Abcam, 70,303), anti-rabbit IgG control antibodies (Millipore, Billerica, MA) and
mouse monoclonal anti-FLAG M2 beads (Sigma–Aldrich) for FLAG-tagged POU3F3
experiments.

ChIP was performed using approximately 1 × 10⁷ N2A cells per assay. Cells were trypsinized, resuspended in 10 ml PBS containing 1% final concentration formaldehyde and incubated for 10 min at room temperature with rotation. Cross-linking reactions were quenched with 0.125 M glycine for 5 min at room temperature and washed twice with ice-cold PBS. Nuclei were then isolated and chromatin was sheared to approximately 500 bp using a Bioruptor (Diagenode). Cross-linked chromatin was immunoprecipitated using 5 μg anti-rabbit PAX6 or anti-rabbit IgG control antibodies (both Millipore) overnight at 4°C. Complexes were collected using Protein-A magnetic beads (Pierce) pre-blocked with BSA (New England Biolabs) and transfer RNA (Roche), then washed and eluted. Cross-links were reversed at 65°C overnight and DNA was precipitated, treated with Proteinase K (Roche) and then purified using a PCR Purification Kit (Qiagen).