GSM1824717
GSM1824717: Limb Mesenchyme Input; Mus musculus; OTHER
Twenty five oligonucleotide probes corresponding to the murine Hotchon transcript were selected using the Stellaris Probe Designer Software set to masking level 5 to ensure the maximal level of probe specificity (biosearchtech.com) (Supplemental File).  The oligonucleotide sequences selected by the software were synthesized with an 18-atom hexa-ethyleneglycol spacer followed by a biotin tag located at the 3’ end of each oligonucleotide and purified by HPLC by Integrated DNA Technologies (Corallville, IA).   The distal half of mouse embryonic limb buds at embryonic day (E) 11 were dissected and digested for 10 minutes in sterile PBS containing 0.1% trypsin and 0.1% collagenase at 37 degrees Celsius as described (Owens and Solursh, 1982).  The digested limb buds were dissociated into a single cell suspension by passing them through a 70 µm nylon mesh and fixed in 1% glutaraldehyde as described (Chu et al., 2011.  Approximately 2000 embryonic limb buds were required to produce the 200mg cells needed for each for each ChIRP assay (Chu et al., 2011).  Cell lysis and chromatin shearing were accomplished using a Bioruptor instrument (Diagenode Denville, NJ) as previously described (Chu et al., 2011).  Once sheared, the chromatin was hybridized with the biotin-tagged Hotchon oligonucleotide probes which were divided into even and odd probe pool sets as described (Chu et al., 2012).  As a control, chromatin from E15.5 murine glial cells (a kind gift from Dr. Peter Hurlin), that do not express the Hotchon, were used in parallel assays and hybridized with the same Hotchon even and odd probe pool sets.   After hybridization the chromatin-Hotchon probe complexes were isolated using magnetic streptavidin beads as described (Chu et al., 2011).  DNA from isolated chromatin was purified using phenol/chloroform extraction followed by DNA precipitation as described (Chu et al., 2011).  The precipitated DNA was quantified using a Qubit 2.0 DNA fluorometer (Life Technologies) and submitted to Elim Biopharm (Hayward, CA) for library preparation and next generation sequencing using an Ilumina HiSeq 2500 sequencer (Illumina San Diego, CA). Libraries were constructed for next generation sequencing using the Illumina library preparation kit as described by the manufacturer.
GEO
GSM1824717
GEO Accession
GSM1824717