OreR_CENPA_ChIP_R2
CENP-A ChIPs were performed using an affinity purified rabbit anti-CENP-A antibody (gift of Gary Karpen) that we previously validated for ChIP using S2 cells that contain LacI/lacO inducible ectopic centromeres (Chen et al., Dev Cell, 2015). ChIP in embryos: Embryo collection, fixation, and chromatin isolation were performed as described in Sandmann et al., Nature Protocols, 2007. We carried out two biological ChIP replicates from independent overnight collections of fixed Oregon R embryos (approx. 1.5g each). Chromatin was sheared to 200-500bp using a Bioruptor sonicator (Diagenode), aliquoted, and flash frozen. For the first biological replicate (ChIP1), we performed two technical ChIP replicates using the MAGnify kit, with 15l of dynabeads, approximately 60g of chromatin in 200l volume, and 3l of anti-CENP-A antibody. The second biological replicate (ChIP2) was performed following the Sandmann et al., Nature Protocols, 2007 using 165g of chromatin in 500l volume and 30l of protein A agarose beads. Libraries were made from eluted DNA using the Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences; Replicates 2 and 3) for ChIP1 and the TruSeq ChIP kit (Illumina) for ChIP2 (Replicates 1 and 5). The libraries were sequenced by paired-end on the NextSeq platform using Reagent v.2.  The quality of the chromatin was confirmed by control ChIPs with 15g of chromatin in 200l volume and 2l of rabbit anti-H3K27Ac (Thermo Fisher). The eluted DNA was analyzed by qPCR confirming enrichment of the Rpl32 promoter (F-TTGTTGTGTCCTTCCAGCTTCA and R-TTGTTGTGTCCTTCCAGCTTCA), and lack of enrichment of Rpl32 5 region (F-GGCACGGCGCCAAAATTAATCA and R-CCGATGCCACTGCCTCTTTGGT (Bonn S. et al. 2012 PMID:22538849).