M1713
DNAseq of Paramecium bursaria Dd1 :MAC
The aposymbiotic strain of Dd1 in the early stationary phase was starved for 2 days and collected by water pipe equipped with 11m pore size nylon membrane (NY1102500, Millipore). Around 2E+06 cells were used to isolate the macronuclei. The cells were washed with modified Dryls solution twice and lysed by equal volume of 0.25 M TCMS buffer (10 mM Tris-HCl pH 8.0, 2 mM CaCl2, 8 mM MgCl2, 0.25 M sucrose) with 0.3% (w/v) NP-40. The extract was sonicated (~100 watts) for 6 mins to dissociate the macronuclei and micronuclei. 7 ml extract was loaded on 8 ml 1.6 M TCMS buffer in a 15 ml falcon tube, and centrifuged at 3000 rpm, 4 , 15 min. The macronuclear pellet was washed with 10 ml 0.25 M sucrose once and centrifuged at 3000 rpm, 4 , 15 min. The precipitation was collected and part of the precipitation was stained with SYTOX Green to calculate the ratio of Mac and Mic. The precipitation with the ratio of number of Mac to the total number of nuclei greater than 0.8 was used. The DNA was extracted by the QIAGEN Genomic-tip 20/G (Cat No.10223, QIAGEN).
M1713
SUB6042154
M1713