ChIP-Atlas: SRX733655

Antigen

Cell type Class: Neural
Cell type: Prefrontal Cortex
MeSH Description: The rostral part of the frontal lobe, bounded by the inferior precentral fissure in humans, which receives projection fibers from the MEDIODORSAL NUCLEUS OF THE THALAMUS. The prefrontal cortex receives afferent fibers from numerous structures of the DIENCEPHALON; MESENCEPHALON; and LIMBIC SYSTEM as well as cortical afferents of visual, auditory, and somatic origin.

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: 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.