LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [NAD-seq]
To investigate NADs associated DNAs in the naïve and primed hES cells. Overall design: In this study, we profiled for DNA loci within the nucleolus associated domain (NADs) in naïve and primed H9 hES cells for comparison. We followed a previously published “crosslinked method of nucleoli isolation” protocol described by Vertii et al. (Vertii et al. 2019). Naïve and primed H9 hES cells maintained in RSeT and mTeSR, respectively, were collected for preparing NADs sequencing sample. 20-30 x 10^6 cells were subjected to ultrasonic cell disruption after crosslinking the cells. Nucleolar fraction was spin pelleted after 2-3 rounds of sonication in the high magnesium (HM) buffer and then subject to another round of sonication and nucleolar pelleting in the low magnesium (LM) buffer. The final pelleted nucleolar DNA were extracted and processed to DNA library preparation with the Illumina`s TruSeq DNA PCR-free Library Preparation kit (Illumina # 20015962), and 350 bp fragments were sonicated and size-selected by sample-purifying beads following manual instruction. The library was subjected to 150bp paired-end sequencing onNovaSeq. The details of protocol is described by by Vertii et al. (Vertii et al. 2019). More protocol details are described in the methods of this study.