GSM8187170: PPARG-KQ_2_input; Homo sapiens; ChIP-Seq
Cell lysis and chromatin fragmentation on cross-linked whole-cells was prepared in ChIP buffer (50 mM Tris-HCL pH 7.5, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaDOC, 0.1% SDS) with protease inhibitors (1 mM PMSF and Complete protease inhibitor tablet from Roche) by probe sonication (Fisher Scientific F60 Sonic Dismembrator). Samples were sonicated at low power (level 4) for three pulses of 10 seconds each and at a higher power (level 6) for two pulses of 10 seconds each. Proteins were bound at 4°C overnight with 10 μg anti-PPARγ (sc-7196, Santa Cruz Biotechnologies) in ChIP buffer with protease inhibitors and then immunoprecipitated with Protein A Sepharose Beads (GE Healthcare). Beads were washed for 5 minutes each with the following buffers: ChIP Buffer, mixed Micelle buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 5.2% Sucrose, 1% Triton X-100, 0.2% SDS), Buffer 500 (250 mM NaCl, 5 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 25 mM HEPES, 0.5% Triton X-100, 0.05% NaDOC), LiCl/Detergent Buffer (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% IGEPAL CA-630, 0.5% NaDOC), and Tris-EDTA pH 8.0. Cross-linking was reversed at 65°C overnight in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and DNA was isolated using phenol/chloroform extraction and NaCl/EtOH precipitation with 20 μg glycogen carriers. Approximately 10 ng of ChIP DNA (quantified by Qubit 2.0 Fluorometer, Invitrogen) was prepared for sequencing according to the amplification protocol from Illumina using enzymes from New England Biolabs and PCR purification (#28104) and MinElute (#28004) kits from Qiagen. Deep sequencing was performed by the Functional Genomics Core (J. Schugg and K. Kaestner) of the Penn Diabetes Research Center using Illumina HiSeq 2000 and aligned sequences were obtained using the Solexa Analysis Pipeline.