Lls were transferred from the flask to a pre-chilled centrifuge tube
Lls were transferred from the flask to a pre-chilled centrifuge tube by scraping gently with a cell scraper. Crosslinked cells were homogenized by douncingSarachana and Hu Molecular Autism 2013, 4:39 http://www.molecularautism.com/content/4/1/Page 5 of40 to 50 times on ice using a dounce homogenizer with a tight pestle to release the nucleus. Optimal cell lysis was assessed under a phase contrast microscope using a hemacytometer. The cell lysate was transferred to a 1.7 ml microcentrifuge tube and centrifuged for 10 minutes at 5,000 rpm (2,400 RCF) in a 4 microcentrifuge to pellet nuclei. Chromatin was then isolated from the nuclear pellets and sheared into 150 to 1,000 bp fragments by incubating with 10 U/ml (final concentration) Enzymatic Shearing Cocktail (Active Motif) at 37 for exactly 10 minutes. The enzymatic shearing reaction was stopped by adding EDTA to a final concentration of 10 mM EDTA and chilling the reaction tube on ice for 10 minutes. To assess shearing efficiency and determine DNA concentration in the sheared chromatin, a 50 l aliquot of each sheared chromatin sample was reverse-crosslinked by mixing with 150 l nuclease-free water and 10 l 5 M NaCl. The reaction was incubated at 65 in a water bath overnight. After incubation, 1 l RNaseA (10 g/l) was added to each tube and the reaction was incubated at 37 for 15 minutes. The reaction was then mixed with 10 l Proteinase K (0.5 g/l) and further incubated at 42 for 1.5 hours. The reverse-crosslinked DNA was isolated using standard phenol/chloroform extraction technique and purified using PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 the Chromatin IP DNA Purification Kit (Active Motif). DNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Optimal shearing was assessed by agarose gel electrophoresis. For chromatin immunoprecipitation reaction, the remaining enzymatically sheared, non-reverse-crosslinked chromatin was aliquoted into multiple tubes, each of which contained approximately 25 g chromatin DNA. Each aliquot of chromatin was then used as input chromatin for sequential immunoprecipitation according to the manufacturer’s protocol for the Re-ChIP-IT Kit (Actif Motif). For each reaction, sheared chromatin (approximately 25 g per reaction) was first immunoprecipitated by mixing with 1 g of anti-AR, anti-ER, anti-RORA, or IgG antibody and 25 l Protein G Magnetic Beads (Active Motif). The reaction was then incubated on an end-toend rotator overnight at 4 . After incubation, the immunoprecipitated chromatin was eluted from the magnetic beads using the Re-ChIP-IT Elution Buffer (Active Motif) and desalted using the Active Motif Desalting Columns to BX795 site remove the first antibody on the chromatin. Then, 1 g of the second antibody (anti-NCOA1, antiNCOA5, anti-SUMO1, anti-FHL2, or IgG antibody) and 25 l of the LSV Protein G Magnetic Beads (Active Motif) were added to the desalted chromatin (approximately 90 l) and incubated on an end-to-end rotator overnight at 4 to re-immunoprecipitate the chromatin. To validate that successful re-immunoprecipitation was caused by the second antibody and not by carried over first antibody, are-immunoprecipitation reaction without the second antibody (that is, no-second-antibody control) was also performed in parallel and included in subsequent qPCR analysis. After incubation, DNA from re-immunoprecipitated chromatin was isolated and purified using the ChIP DNA Purification Kit (Active Motif). The list of antibodies for sequentia.