A Phylogenetic Study of Cycads (1915)(en)(5s) by Chamberlain C.J.

By Chamberlain C.J.

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Allow the frozen cell pellet to thaw on ice. Using your fingers, flick the bottom of the tube sharply to evenly disperse the cells in the pellet (see Note 3). 2. Resuspend the cells in 8 ml of cold Buffer 1 and incubate for 10 min on ice. Mix intermittently by gently inverting the tube. 40 M. Murtha et al. 3. Transfer the cell suspension to a glass dounce (that had been kept cold on ice), and dounce the cells ten times using a B pestle. 4. Transfer equal portions of the dounced cell suspension into two 15 ml conical polypropylene tubes and pellet the cells at 2K rpm, 7 min at 4°C using a tabletop centrifuge.

A single DHS site surrounded by heterochromatin is shown at the top. The ovals represent highly packed nucleosomes, and the arrows are DNase I cleavage sites, which occur much more frequently in DHS regions. Limited DNase I digestion releases short DNA fragments from DHS regions (red arrows, and red fragments), as well as larger fragments that result from random cutting of genomic DNA (blue arrows, and blue fragments). The short DNA fragments are then separated from larger fragments by sucrose gradient ultracentrifugation, as shown in the gel at the center.

4. Remove each liver and rinse in ice-cold PBS. , for RNA isolation at a later time. 5. 15% KCl. 15% KCl. 6. Carefully dry off the excess KCl solution with a Kimwipe. Weigh the liver and submerge the minced liver in ice-cold NEHB buffer. 2. Tissue Homogenization and Isolation of Nuclei (Scale: Pool of Three to Four Mouse Livers) (See Note 1) Isolation of Nuclei for DNase-seq 17 1. Homogenize the liver in a 30 ml Potter-Elvehjem Tissue Grinder with three to four strokes. Use a buffer-to-tissue ratio of ~6 (v/w) (see Note 3).

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