S2_CENPA_Input
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). Chromatin from 106 fixed Drosophila Schneider (S2) cells (approx. 90g) were used for each IP and chromatin was sheared to 100-300bp using a Covaris sonicator. ChIP were performed using the MAGnify kit (Thermo Fisher). 1l of the anti-CENP-A antibody was coupled to 10l of beads for 2hrs followed by incubation with chromatin overnight at 4oC. DNA was eluted in 50l of elution buffer. Libraries were generated using the TruSeq kit (Illumina) and paired-end sequenced using the Reagent kit v.3. (Illumina) on the NextSeq platform 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).