title: MaPTOL for REST
status: Public on Apr 01 2008
contact.city: San Diego
contact.first: Olivier
contact.last: Harismendy
contact.state: CA
contact.address: 10550 North Torrey PInes Road
contact.department: Neurobiology
contact.country: USA
contact.institute: The Scripps Research Institute
organism: Mus musculus
manufacture_protocol: Chromatin was prepared from mouse E12.5 embryos, sheared by sonication, and precleared with Protein-G agarose beads according to {Weinmann, 2002 #6}. An antibody (REST-H290 Ð Santa Cruz) targeted against the N-terminal region of mouse REST was used for immunoprecipitation under standard ChIP conditions {Weinmann, 2002 #6} with the following modifications. Two rounds of immunoprecipitation were carried on successively. During the second ChIP, after washing the bead-chromatin complex, they were equilibrated in NEB restriction enzyme buffer 4 with 0.5 % NP40 to sequester SDS. 80 µl of beads-chromatin slurry were then digested for 6hrs with 80 units of NlaIII. This step facilitates cloning of the co-immunoprecipitated DNA fragment and also provides footprint information as proteins that are bound to the DNA may prevent cleavage. After digestion, the complexes were again washed, eluted, and crosslinks reversed according to standard protocols {Weinmann, 2002 #6}. The entire eluate was cloned into the tagging vector pTagBM that contains two head to head MmeI restriction sites flanking the SphI cloning site. This initial library was amplified in bacteria (ECLONI Ð Lucigen) and purified using Invitrogen MaxiPrep Filter kit. 5 µg of this library was digested by 192 U of MmeI restriction enzyme overnight. After inactivation of the enzyme, we blunted the cut plasmid by incubation with 6U of T4 DNA polymerase and 10 pmol of dNTP during 3hrs at 14¼C. We stopped the reaction by addition of 10 mM EDTA for 20 min at 75¼C. The cut library was then purified by separation on an agarose gel and gel extraction using Qiagen Gel Extraction kit. 50 ng of the library was religated by overnight incubation at 14¼C with 1 µl of Invitrogen T4 DNA ligase. The religated library was used to transform E. coli competent cells (ECLONI Ð Lucigen). After quality control for religation by PCR, the library was amplified by growth on LB agar dishes (25x25 cm) and purified using Invitrogen MaxiPrep Filter kit. 100 µg of the library was digested overnight by 500 U of BamH1 restriction enzyme which sites encompass the two Mme1 sites on the pTagBM vector. The MaPTags extracted during this process were purified on an 8% polyacrylamide-TBE gel and electro-eluted from the gel (BioRad). 10 µg of MaPTags were subjected to sequencing.