GSM1526505: S10.H3K4me3.NeuN_negative; Homo sapiens; ChIP-Seq
Tissue aliquots (200–500 mg/subject) were extracted in hypotonic lysis buffer, purified by ultracentrifugation and resuspended in 1x PBS, immunotagged with anti-neuronal nucleus (anti-NeuN, Millipore) antibody and sorted into NeuN(+) and NeuN(-) fractions. Mononucleosomal preparations from at least 1×106 sorted nuclei were prepared for subsequent chromatin immunoprecipitation with anti-H3K4me3 antibody (Upstate/Millipore) ChIPed DNA was first blunt-ended by End-it DNA Repair Kit (Epicentre) at RT for 1hr. Reaction was cleaned by column (Qiagen), and DNA was A-tailed by exo- Klenow DNA Polymerase (Epicentre) at RT for 1hr. Reaction was again cleaned by column, and DNA was ligated to Genomic Adpater Oligo Mix (Illumina) by Fast-link DNA Ligation Kit (Epicentre) at RT for 2 hours followed by 16°C O/N. Reaction was cleaned by column, and ligated DNA was amplified using PfuUltra II Fusion HS DNA Polymerase (Stratagene) with 250µM dNTPs and 1µL of each of the Genomic PCR Primers (Illimina) in 100µL volume with the following cycling conditions: 95°C 1min; 15 cycles of 95°C 50sec, 65°C 1min, 72°C 30sec; 72°C 5min. PCR product was cleaned and volume reduced by column before loading onto a 1.8% agarose gel. Typically a strong smear of 160-230bp (including 92bp from the Illumina primers) was observed.
GEO Accession
GSM1526505