title: GSM9168088: aorta, IgG control; Mus musculus; ChIP-Seq
library_name: GSM9168088
library_construction_protocol: For PRDM16 ChIP in C57Bl6/J aortae, 12 were dissected, dual-crosslinked with 1.5mM EGS for 20 min followed by 1% formaldehyde for 10 min, then washed and snap frozen in liquid nitrogen.  Aortae were pooled into a 2mL safe-lock tube with a 3mm stainless steel bead and cryo-milled into a powder using 2 cycles of 5Hz for 30 sec and 23 Hz for 30 sec.  The pulverized aortas were placed on wet ice, allowed to thaw slightly and then resuspended in 0.6% SDS lysis buffer.  Chromatin was sheared using a microtip probe sonicator at 25% Amplitude, 30 sec on, 20 sec off for 5 minutes.  After the first minute of shearing the insoluble ECM clump was removed with a pipet tip and shearing resumed for the remaining 4 minutes.  Dilution buffer was added to make a 0.1% SDS final IP concentration and the sample was split into 3 separate immunoprecipitations with approximately 10ug chromatin per IP. ChIP DNA was purified using Active Motif Chromatin IP Purification Kit and used for downstream library prep. Sequencing libraries were made according to manufacturer's recommendations using the Roche KAPA HyperPrep Kit and Roche Universal Dual-Index Adapters.  Size selection was performed using Beckman Coulter SPRI Select beads for an 800 bp upper limit cut-off