Cell nuclei were isolated, pelleted, and sonicated.
The isolated lymphocyte pellet and urine samples were stored frozen at –80°C and hand-carried frozen on dry ice to New York University.
The isolated pectin pellet was washed twice with cold ethanol, and the solid material was dried at 60°C in an air-forced oven.
A fraction of the isolated nuclear pellet was fixed in 4% paraformaldehyde in 1 × PBS to determine the intactness of nuclei by staining with 4',6-diamidino-2-phenylindole (DAPI).
After washing five times with 20 volumes of washing buffer (50 mM Hepes-KCl, pH 7.9, 100 mM KCl, 3 mM MgCl2), the RNAs were isolated from pellet fractions.
Cells were manually isolated or pelleted from the culture medium.
The genomic DNA and the total RNA were isolated from pellets containing 5E6 cells on an automated QIAcube workstation.
For sonication-based 4C library preparation, the isolated chromatin pellets were re-suspended in SDS lysis buffer (1% SDS, 5 mM EDTA pH=8.0, 50 mM Tris HCl pH=8.0, 1x protease inhibitor cocktail) and sonicated to an average size of 500 bp.
From each strain, membrane vesicles were isolated and pelleted as an insoluble fraction of the cell lysate according to the method of Azuma et al. They were suspended in a small volume of a buffer comprising 50 mM Tris-acetate (pH 8.0), 1 mM DTT, 2% glycerol and 250 mM sucrose in order to isolate cytoplasmic membrane as follows.
All exosomes samples were tested for endotoxin contamination using Limulus Amebocyte Lysate (LAL) test (Catalog number KT05, Houshiji, Co. Ltd .. Freshly isolated exosome pellets were resuspended and fixed in phosphate buffer containing 2% glutaraldehyde and then loaded on Formvar/carbon-coated electron microscopy grids.
