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 – 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 – 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 14ºC. We stopped the reaction by addition of 10 mM EDTA for 20 min at 75ºC. 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 14ºC with 1 µl of Invitrogen T4 DNA ligase. The religated library was used to transform E. coli competent cells (ECLONI – 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.
Description
not applicable
Data processing
The short sequence reads were processed automatically and sequences flanked by two CATG motifs were extracted. MaPTags with a length between 30 to 36 nucleotides (variation that is inherent in MmeI digestion) were retained for the analysis. MaPTags were then consolidated with their reverse complement and grouped by unique sequences.
The first 14 nucleotides of each MaPTag indexed after the CATG were used to search the Mouse Genome database (UCSC mm8). Only perfect matches were accepted. If a hit was found, then a match to the last 14 nucleotides was sought by searching up to 3kb downstream of the first hit, on the same strand in the opposite orientation. If both halves of a MaPTag were found, the junction between the 2 halves of the tag was then compared to the genome sequence. If the junction region also matched (with up to 1 mismatch allowed), then the tag was considered a hit and the location was defined as a MaPTag locus. MaPTags with slightly different sequences but the same location (due to variation in the position of Mme1 digestion within the same cloned fragment), were consolidated into one locus. All of the loci are associated with their count number, number of intervening CATG in the locus, and the percentage of each half tag located in a repeated region.
