GSM8147310: MNase wt P19 ECCs +RA, rep2; Mus musculus; MNase-Seq
For MNase-seq assays, 10-20.106 cells were incubated for 10 min in 1% formaldehyde in PBS, washed twice and scraped in ice cold PBS and spun down at 100g for 10min at 4°C. Cells were re-suspended in digestion buffer (50 mM Tris-HCl pH7.6, 2 mM CaCl2, 0.2% NP40) containing protease inhibitors (complete EDTA-free, Roche) and warm-up to 37°C for 2min. 4U of MNase were added to wt ECC chromatin and the reaction mixture was incubated at 37°C for 5 min. For TDG-null ECCs, 2U of MNase were added, due to the limited amount of recovered mono-nucleosomal DNA when using 2U of MNase. The reaction was stopped by adding EDTA to a final concentration of 10mM. Cells were then incubated for 10 min on ice in SDS lysis buffer (1% SDS, 50 mM Tris pH8, 10 mM EDTA pH8). An equal volume of TE/RNase (10 mM Tris pH8, 0.1 mM EDTA pH8, 0.4 µg/ml RNase A) was added before incubating at 37°C for 2 hours. A Proteinase K digestion (1 mg/ml) was then performed at 55°C overnight. The mixture was then extracted twice with Phenol/chloroform and the DNA was precipitated 1 hour at -20°C in the presence of glycogen (20µg).  After centrifugation, DNA pellets were washed by ethanol, dissolved in TE buffer (10 mM Tris pH8, 1 mM EDTA pH8) and purified using NucleoSpin Gel and PCR clean-up (Macherey-Nagel). DNA was loaded on a 2% agarose gel. The mono-nucleosome band was excised and the corresponding DNA was purified using MinElute Gel Extraction kit (Qiagen). Mono-nucleosomal DNA was further analyzed by deep sequencing. MNase-seq libraries were prepared using the TrueSeq ChIP Sample Prep kit (Illumina) and single-end sequenced using a HiSeq 1500 sequencing system.