GSM3771303: MINCE_EdU_Pulse_1; Mus musculus; OTHER
MINCE-seq: 1) Chromatin Preparation after trypsinisation, 120 × 106 cells per sample were fixed with 1% formaldehyde (Thermo, 28908; 10 min at room temperature with occasional mixing) and quenched with 0.125M Glycine (5 min). Fixed cells, after being washed twice with PBS,  were lysed for 10 min on a rotating wheel at 4oC in 5 mL of lysis buffer (50mM HEPES pH 7.8/ 150mM NaCl/ 0.5% v/v IGEPAL/ 0.25% v/v Triton-X/ 10% v/v Glycerol) supplemented with 1× protease inhibitor cocktail (PIC-Roche, 04 693 116 001). After centrifugation, the cells were resuspended in 4 ml Wash Buffer (WB: 10mM Tris-HCl pH 8/ 200mM NaCl)  supplemented with 0.5mM DTT and incubated for 10 min on a rotating wheel at 4o C. Nuclei were pelleted for 5 min at 3000 rpm at 4°C  and resuspended in 1.5 ml RIPA buffer (10mM Tris-HCl pH 8/ 140mM NaCl/ 1% Triton-X/ 0.1% Sodium-Deoxycholate/ 0.1% SDS) freshly supplemented with 1× protease inhibitor cocktail  and 1mM CaCl2.  2) MNase Digestion Samples were equally split in 3 (1.5 ml tubes) and incubated for 10 min in a water bath at 37oC. MNase (Thermo Scientific EN0181) was diluted at 80U/μl in RIPA buffer freshly supplemented with 1× protease inhibitor cocktail and 1mM CaCl2. The digestion was carried out by adding MNase at a final concentration of 6U per million cells and incubation for 5 min at 37oC with occasional mixing. Digestions were stopped by placing tubes on ice and by adding 2× STOP Buffer (2% Triton/ 0.2% SDS/ 300mM NaCl/ 10mM EDTA). Digested chromatin was recovered by overnight rotation at 4oC followed by centrifugation at 4°C  at maximum speed for 10 min. 3) DNA Extraction The chromatin was equally distributed in 1.5 ml tubes and the crosslinking reverted overnight at 65°C in the presence of 1% SDS and shaking (800 rpm). The next day the samples were incubated for 2h at 56°C with 5 μl Proteinase K (GEXPRK01B5 Eurobio) and processed through phenol/chlorophorm extraction and ethanol precipitation. DNA was resuspended in a total volume of 0.4 ml Ultra Pure Nuclease free water. RNase digestion was carried out with 5 μl RNase A (EN0531 Thermo Scientific) for 2 hours at 37°C . The DNA was purified and precipitated again and resuspended in 51 μl Ultra Pure Nuclease free water. 1 μl of DNA was further diluted and the fragment sizes were analysed with a D1000 HS Screentape (Agilent 5067-5584) on an Agilent Tapestation. 4) CLICK-reaction streptavidin pull-down each DNA sample (50 μl) was supplemented, in order, with the following buffers: 342.5 μl of 100 mM Potassium Phosphate Buffer (15433339 Fisher Scientific); 50 μl of 10 mM Biotin-TEG Azide (762024 Sigma ) resuspended in DMSO; 7.5 μl of pre-mixed Cupric Sulfate Pentahydrate (2.5 μl at 20mM; C8027 Sigma) and THTPA (5 μl at 50 mM; 762342 Sigma); 25 μl of 100 mM Aminoguanidine Hydrochloride (396494 Sigma); 25 μl of 100 mM Sodium Ascorbate (A4034 Sigma). After briefly vortexing, the reactions were performed for 1.5 hours at room temperature protected from light, and  DNA was subsequently ethanol-precipitated and resuspended in 400 μl ultra pure water. Twenty microliters were kept as input and the rest (380 μl) were used for streptavidin pull down. For this, 25 μl M-280 streptavidin magnetic beads (10292593 Thermo Scientific) per reaction were washed 3 times (10 min rotation at room temperature per wash) with 1 ml 1× Wash and Binding buffer (W&B; 2×:  10 mM Tris-HCl pH 7.5; 1 mM EDTA; 2 M NaCl; 0.2% Tween 20) and resuspended in 25 μl 1× W&B buffer. EdU-labelled DNA was then pulled down with 380 μl 2× W&B buffer and 25 μl washed beads (1 hour at room temperature on a rotating wheel). Beads were separated by placing the tubes on a magnetic rack and the unbound fraction was stored at -20oC. The beads were washed 3 times with 1ml W&B buffer (10 min at room temperature on a rotating wheel). Washed beads were resuspended in 100 μl TE/1% SDS and DNA was eluted with 20 μl Proteinase K (1 hour at 65oC with occasional vortexing). After adding TE to a final volume of 200 μl, the DNA was phenol/chloroform-extracted, ethanol-precipitated and resuspended in 20 μl ultra pure water.