GSM5261293: ucMSC - Pol2 - Old - Batch1; Homo sapiens; ChIP-Seq
Approximately 1 million PD12 and PD32 hMSCs were fixed in 1% formaldehyde at room temperature for 10 minutes. Following 3 washes in cold PBS, cell pellets were flash frozen on dry ice and store at -80ºC. Samples were thawed on ice and resuspended in 100uL RIPA with 10uM PFMS and 1× Halt Protease Inhibitor Cocktail (Thermo Fisher PI78439). Samples were sonicated to release chromatin with an average fragment length of 400bp in an EpiShear Multi-Sample sonicator (Active Motif 53062) using the following parameters: 5 minutes sonication, 30 second ON/OFF cycles, 50% amplitude at 4ºC. For each ChIP, 10uL of clarified lysate was added to 90uL of HBSS and used as input into the True MicroChIP Kit (Diagenode, C01010130); manufacturer's instructions were followed beyond the fixation/sonication part of the protocol, using 2ug of each antibody. 2uL of clarified lysate was used as input and was treated as a sample starting from the reverse crosslinking step in the protocol. The ChIPed DNA was cleaned and concentrated using the Qiaquick PCR Purification Kit (Qiagen, 28104), following the manufacturer's protocol with the following exception Sequencing libraries were prepared using the MicroPlex Library Preparation Kit v2 (Diagenode, C05010014) following manufacturer's instructions
GEO
GSM5261293