LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [pA-DamID LAD-seq]
To investigate LADs associated DNAs in the naïve and primed hES cells. Overall design: In this study, we profiled for DNA loci within the nucleus Lamina associated domain (LADs) in naïve and primed H9 hES cells for comparison. We followed a previously published pA-DamID sequencing method using LMNB1 antibody. So, Genes in close contact with LMNB1 are DamID labelled (methylated) and thus annotated via sequencing. Naïve and primed H9 hES cells maintained in RSeT and mTeSR, respectively, were collected for pA-DamID reaction. The experiment was performed using the rabbit antibody against LMNB1 (Abcam # ab16048) to tag pA-Dam fusion protein to LADs and conducted to DamID sequencing onwards. The details of protocol is described by van Schaik et al. (van Schaik et al. 2020) for the pA-Dam step, and by Vogel, Peric-Hupkes, and van Steensel (Vogel, Peric-Hupkes, and van Steensel 2007) for the DamID sequencing step. More protocol details are described in the methods of this study. The DamID-DNA library was subjected to 100bp single-end sequencing on HiSeq 2500 with 1%PhiX spike-in.