Human, ChIP, Cortex, GW23, TCF4
Tissues were mechanically dissociated in ice-cold HBSS and fixed in 1% PFA for 10 min at RT. Fixed cells were lysed in a hypotonic buffer. Nuclei was extracted by centrifugation at 13,500 rpm for 10 min at 4°C and sheared into 200 - 1,000 bp fragments using a Covaris S2. Immunoprecipitation reactions were were performed by diluting the sheared chromatin 1:10 in ChIP dilution buffer (16.7 mM Tris-HCl pH=8.0, 1.2 mM EDTA, 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100) in 3 mL final volume. 100 uL was removed as “input”. 5 ug of primary antibody against each 5 TRs were added to each IP. ChIP DNA was purified using ChIP DNA Clean and Concentrator kit (Zymo Research,D5205). ChIP-seq libraries were generated using Ovation Ultralow System V2 Multiplex System (NuGEN) following manufacturer's protocol, using 12 PCR cycles. The resulting libraries were size selected 180–350 bp using BluePippin (Sage Science) and sequenced at the Center for Advanced Technology at UCSF (Illumina HiSeq 4000; http://cat.ucsf.edu/) using a single read 50-bp strategy.