Sorted cells were crosslinked by addition of 1/10th volume of fresh 11% formaldehyde solution. Before ChIP, cells were resuspended, lysed, and sonicated to solubilize and shear crosslinked DNAs. The resulting whole-cell extract was incubated LHX2 antibody. After ChIP, samples were washed with low salt, high salt, LiCl, and Tris-EDTA buffer. Bound complexes were eluted and crosslinking was reversed. Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.  Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 25 cycles and library fragments of 150-300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
GEO Accession
GSM1167212