Single-end sequencing of macronuclear DNA from Nowa1 knockdown
Post-autogamous cells starved for 4 days were filtered through several layers of sterile gauze and collected by low-speed centrifugation (250× g for 1 min) and washed in 10 mM Tris-HCl pH 7.4. The cell pellet was diluted 5-fold by addition of lysis buffer (0.25 M sucrose, 10 mM MgCl2, 10 mM Tris pH 6.8, 0.2% Nonidet P-40) and processed as described in (Preer et al., 1992). All steps were performed at 4°C. 1 ml of resuspended cells was lysed with 20-30 strokes of a Potter homogenizer, followed by the addition of washing buffer (0.25 M sucrose, 10 mM MgCl2, 10 mM Tris pH 7.4 to a final volume of 10 ml. Developing new macronuclei were collected by centrifugation at 600g for 1 min and washed 3 times in washing buffer. The pellet was diluted in washing buffer, loaded on top of a 3-ml sucrose layer (2.1 M sucrose, 10 mM MgCl2, 10 mM Tris pH 7.4) and centrifuged in a swinging rotor for 1 h at 210,000g. The nuclear pellet was collected and diluted 5-fold in 10 mM MgCl2 10 mM Tris pH 7.4 prior to addition of two volumes of proteinase K buffer (0.5 M EDTA pH 9, 1% N-lauryl sarcosine sodium, 1% SDS, 1 mg/ml proteinase K). The lysates were incubated at 55°C for at least 12 h, gently extracted with phenol, and dialysed twice against TE (10 mM tris-HCl, 1 mM EDTA, pH 8.0) containing 20% ethanol and once more against TE.