Sequencing of DNA from the biotinylated chromatin from HeLa S3 clonal cell line

Sequencing shows that macroH2A1-emerin interaction occurs in lamina-associated domains on a genome-wide scale. We decided to verify that the biotinylated signal is indeed enriched in the chromatin domains positioned at the nuclear periphery. In this regard, we purified the biotinylated chromatin from HeLa S3 clonal cell line stably coexpressing BirA-emerin and BAP-macro-H2A1 using proximity utilizing biotinylation native chromatin immunoprecipitation (PUB-nChIP). Instead of analysing the protein fraction, we isolated DNA from the biotinylated chromatin pull down and performed high throughput sequencing in order to get insight into the genome wide distribution of the isolated DNA. Importantly, PUB-nChIP-seq of the DNA purified from HeLa S3 clonal cell line stably coexpressing BirA-emerin and BAP-macro-H2A1 showed high levels of enrichment at the lamina associated domains, which were identified previously in HeLa cells using anti-Lamin B1 and anti-Lamin A chIP-seq (Lund et al., 2015). The enrichment of BirA-emerin labelled BAP-macro-H2A at LADs is visibly clear at both the chromosome level and at individual LADs. To test if this enrichment was significant, the genome was divided into 100kb windows and monte carlo simulation using 10,000 iterations was performed to examine if probes overlapping LADs were significantly different to the genomic average. For both replicates, at both LMNA and LMNB1 LADs, the enrichment of biotinylated BAP-macroH2A1 observed was significant relative to the genome wide average. Total (nonbiotinylated) and biotinylated chromatin from Hela S3 cell line stably coexpressing BirA-emerin and BAP-macroH2A1 fusion proteins was isolated. The chromatin was digested with Mnase. Input DNA from the total chromatin was isolated and sequenced. For library construction, the DNA fragments (100 ng) were converted to blunt ends by end repair using Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific) and ligated to Ion Xpress Barcode Adapters.