Pcr purification kit columns

ChIP elution buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 1% SDS) was added to each nuclei aliquot to a final 100 µL volume. Samples were de-crosslinked and deproteinized by adding 4 µL of 5 M NaCl, 2 µL 10 mg/mL DNase-free RNase A and 2 µL of 20 mg/mL proteinase K and incubating 30 min at 37 °C followed by 65 °C incubation for a minimum of 2 h. Samples were purified using Qiagen PCR purification kit columns. DNA was quantified using a Qubit DNA HS kit and fragment size distribution monitored by capillary electrophoresis. Cell number for tissue samples was estimated from the total DNA amount, taking into account that each mouse diploid cell contains approximately 6.6 pg of DNA.

ChIP assay was performed via a commercially purchased chromatin immunoprecipitation kit (Millipore, Temecula, CA), using anti-POU2F1 or anti-IgG antibodies. Cells were first cross-linked for 10 min by adding formaldehyde directly to culture medium to a final concentration of 1%. Cross-linked cells were then washed twice with cold PBS (with protease inhibitors), scraped, pelleted, resuspended in 200 μL SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.0), and incubated for 10 min on ice. The lysates were then sonicated for five cycles of 30 s each, resting on ice for 1 min between cycles. After sonication, the samples were centrifuged and the supernatants diluted tenfold in ChIP dilution buffer with protease inhibitors. Cross-linked chromatin was incubated overnight with 5 μg anti-POU2F1 or anti-IgG of 1 mL at 4 °C. Antibody-protein-DNA complexes were isolated by immunoprecipitation. After extensive washing, pellets were eluted by freshly prepared elution buffer (1% SDS, 0.1 M NaHCO₃). Formaldehyde cross-linking was reversed by 5–12-h incubation at 65 °C after adding 20 μL 5 M NaCl. Samples were purified through PCR purification kit columns (Qiagen, Chatsworth, CA) and used as a template in PCR. The primer sequences are listed in Additional file 1.