ChIP Assay for Plasma Cell Epigenetics

ChIP was performed on DMSO or UNC1999 treated cell lines and CD138⁺ purified plasma cells using a modified version of the OneDay ChIP kit (Diagenode, Liège, Belgium) as previously described [18 (link)]. Chromatin was crosslinked with 1% formaldehyde for 10 minutes at room temperature, followed by 5 minutes treatment with 1 M glycine to stop crosslinking. Chromatin was sonicated (30sec ON/30 sec OFF) for 6 cycles of 5 minutes each at ultrasonic wave output power 320 W in Bioruptor® (Diagenode, Liège, Belgium). Cells were collected and lysed on ice in RIPA extraction buffer with a cocktail of protease inhibitors. Antibodies used are listed in Supplementary Table 4. Precipitated DNA was analyzed by Real Time-qPCR using Platinum® SYBR® Green qPCR SuperMix UDG with Rox (Invitrogen, Carlsbad, CA) and 0.25 mM of each forward and reverse primers. Primer sequences used in this study were, miR-125a: forward primer: 5´-CCCTTCCTCCAGAGCATGAC-3` and reverse primer: 5´-CCATCGTGTGGGTCTCAAGG-3´; miR-320c2: forward primer: 5´-GTTGAGGAGCACTGGGTATGT-3´and reverse primer: 5´-CTTACCCTCTCAACCCA GCTT-3´. The PCR conditions were: 95°C for 2 min followed by 40 cycles of 95°C for 0:30 min and 60°C for 1 min. The run and analysis were performed using Mx3005P instrument and software (Stratagene).