Chromatin Immunoprecipitation to Detect H3K9me3 and H3K27me3

Ventral midbrains were dissected and pooled from 7 embryos treated with HA or vehicle. Tissue was maintained in ice-cold PBS before crosslinking, which was carried out for 10 min, with 1% of formaldehyde. The crosslink reaction was stopped for 5 min with 125 mM glycine and the crosslinked tissue was washed three times with ice-cold PBS for 5 min. After the last wash, tissue was lysed with 500 μL of lysis buffer. Chromatin was sonicated using an Ultrasonic Processor (GENEQ, GEX500; SOVC505-00). After sonication, DNA was extracted and its length evaluated. Quantitation of chromatin was made by the Lowry reaction and 100 μg of material was used per IP. Immunoselection was performed according to the protocol of the OneDay ChIP kit (Diagenode, Kch-oned IP- 180), with anti-H3K9me3 or anti-H3K27me3 (Diagenode). Immunoprecipitation was performed by incubating 20 μL of magnetic beds (16-663 | Magna ChIP^TM Protein A+G Magnetic Beads), with the antibody-chromatin complexes for 3 h in a rotating wheel; then the beads-antibody-chromatin complexes were washed three times with ice-cold ChIP buffer 1×. Finally, the DNA was decrosslinked overnight and purified employing the MinElute Reaction Cleanup Kit (Cat No./ID: 28204) following the manufacturer’s instructions. ChIP assays were performed by qPCR in a fast optical 96-well qPCR reaction plate (Applied Biosystems) with the same primers used for MeDIP/hMeDIP for Pitx3 and Th. The qPCR reaction was performed using Thermo Maxima SYBR Green/ROX1 PCR Master Mix (Thermo Fisher Scientific) with a StepOnePlus Real-Time PCR System (Applied Biosystems), according to the manufacturer’s instructions.