We lysed batches of ~9×109 cells by completely resuspending frozen cell pellets in 25 ml ice-cold urea-based cell hybridization buffer (25 mM Tris pH 7.5, 500 mM LiCl, 0.25% dodecyl maltoside, 0.2% sodium dodecyl sulphate, 0.1% sodium deoxycholate, EDTA 5 mM, TCEP 2.5 mM, and 4 M urea). Next, the cell sample was passed ~10 times through a 27-gauge needle attached to a 25 ml syringe in order to disrupt the pellet and shear genomic DNA. At this point lysates were kept at −80°C or incubated at 65°C for 10 min before clearing by centrifugation for 10 min at 10,000 × g.

For the first purification, frozen cell pellets were resuspended in 8 ml ice-cold detergent-based cell lysis buffer (10 mM Tris pH 7.5, 500 mM LiCl, 0.5% dodecyl maltoside, 0.2% sodium dodecyl sulphate, 0.1% sodium deoxycholate, 1× Protease Inhibitor Cocktail EDTA-free, and 900 U of Murine RNase Inhibitor) (New England Biolabs), and cell sample passed 10–15 times through a 27-gauge needle attached to a 25 ml syringe in order to disrupt the pellet and shear genomic DNA. The samples were then treated for 10 min at 37°C adding 1× DNAse salt solution (2.5 mM MgCl2, 0.5 mM CaCl2), and 900 U of DNase I [Roche] to digest DNA. Samples were returned to ice and the reaction was immediately terminated by the addition of 16 ml 1.5× cold hybridization buffer to stop reaction.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.