GSM6398703: Y1S5; Mus musculus; ChIP-Seq
50mg of 4 young and 4 old mouse liver tissues were used. 5million of nuclei were extracted using the same process described for the ATAC-seq experiment. CUT&RUN  was performed on 1 million nuclei per sample as previously described with modifications (Skene & Henikoff, 2017). Nuclei were prepared using the Nuclei EZ prep kit (Sigma-Aldrich, NUC-101). Subsequently, nuclei were washed twice with Wash Buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine) and bound to activated Concanavalin A beads for 10 min at room temperature. Cell-bead suspension was then resuspended in antibody buffer (20 mM MHEPES, 150 mM NaCl, 0.5 mM Spermidine, 0.05% Digitonin, 2 mM EDTA) and incubated overnight at 4°C with S2-phospho (ab5095), S5-phospho (ab 5408) and PolII-CTD (ab26721) antibodies from Abcam, in a concentration 1:250. Cell-bead suspension was then washed twice with Digitonin Buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, 0.05% Digitonin) and incubated with 2.5 µl EpiCypher CUTANA pAG-MNase, for 10 min. After washing samples twice with Digitonin buffer, 1 µl 100 mM CaCl2 was added to samples which were incubated for 2 h at 4°C, rotating. To stop the reaction, STOP buffer (340 mM Nacl, 20 mm EDTA, 4 mM EGTA, 0.02%Digitonin, 50 µg/ml RNase A, 50 µg/ml Glycogen, sonicated yeast DNA as spike-in) was added in each tube. Samples were incubated at 37°C for 10 min at 500 rpm, and after centrifugation, liquid was collected, DNA was purified using Zymo DNA clean concentrator kit. Libraries were prepared as described in Tessarz et al, 2014