GSM3559633: BG_e11.5_Input; Mus musculus; ChIP-Seq
Transcription factor ChIP was performed using rabbit DLX1, DLX2 and DLX5 antibodies described in the paper. Basal ganglia (consisting of LGE, MGE and CGE progenitor and mantle zones) were dissected from e13.5 CD1 embryos, fixed in formaldehyde and lysed with a hypotonic buffer to obtain the nuclei; these were then lysed in 1% SDS buffer and the chromatin was sheared into 300-1000 bp fragments by sonicating using a bioruptor (Diagenode). Immunoprecipitation (IP) reactions were performed with the sheared chromatin diluted 1/10 times with dilution buffer usually in 6 ml.  Antibody was then added: 5 µg DLX specific Ab. Negative control CHIP reactions used either rabbit IgG (5µg) or blocking peptide (DLX antigen used for immunizing rabbits; 50x molar excess).  Antibody/chromatin complexes were purified using Dynabeads (Invitrogen) and washed extensively in wash buffer. Complexes were eluted, reverse crosslinked, treated with RNase  and Proteinase K and cleaned using a ChIP DNA Clean & Concentrator kit (Zymo Research). The chromatin was quality controlled (QC) using qPCR to check for enrichment of genomic DNA fragments that were expected, and not-expected, to have DLX binding.  Native histone ChIP was performed starting with ~250,000 nuclei from WT and Dlx1/2-/- E13.5 basal ganglia. Nuclei were extracted and digested with micrococcal nuclease (MNase, Sigma). A population of mono- and di-nucleosomes were used in Chromatin immunoprecipitation assays. Antibodies used were specific to H3 monomethyl lysine-4 (H3K4me1), H3 trimethyl lysine-4 (H3K4me3), H3 trimethyl lysine-27 (H3K27me3), and H3 acetylated lysine 27 (H3K27ac). Immunoprecipitated DNA was washed, isolated and cleaned as for the TF ChIP-Seq described above. Libraries were prepared using an Ovation Ultralow DR Multiplex System (Nugen), size selected in the range of 300 bp on a chip from BluePippin (Sage Science) and lastly QC tested on a Bioanalyzer (Agilent). The libraries were sequenced as single 50-nucleotide reads on a HiSeq4000 (Illumina) at Center for Advanced Technology (UCSF).
GEO Accession
GSM3559633