GSM8720660: CUTandTag_7860_PAX8ko2_H3K27ac; Homo sapiens; OTHER
CUT&Tag was performed according to the protocol published by Kaya-Okur et al. (DOI: 10.1038/s41467-019-09982-5) with modifications based on the protocol by Bartosovic et al. (DOI: 10.1038/s41587-021-00869-9). In short 250,000-350,000 cells were lysed and resuspended in antibody buffer with 1:50 diluted primary antibody (monoclonal rabbit anti-HIF-1β antibody [5537S, Cell Signaling Technology] or polyclonal rabbit anti-HIF-1β antibody [NB100-110, Novus Biologicals] or monoclonal rabbit anti-H3K27ac antibody [ab177178, Abcam] or polyclonal rabbit anti-HIF-1α antibody [Cay10006421, Cayman Chemicals] or polyclonal rabbit anti-HNF-1β antibody [HPA002083, Atlas Antibodies, Sigma Aldrich]). Cells were washed and incubated with Dig-wash buffer containing 1:50 diluted secondary antibody (ABIN101961, Guinea Pig anti-Rabbit IgG antibody, Antibodies-Online). Cells were washed again and resuspended in Dig-300 buffer with 1:100 diluted protein A–TN5 transposase (C01070002, Diagenode, loaded with mosaic adapters according to manufacturer's instructions). Following Tn5 loading cells were washed and resuspended in tagmentation buffer. Tagmentation was stopped by addition of 500 mM EDTA, SDS 10% and Proteinase K. DNA was purified using the DNA Clean and Concentrator-5 Kit (Zymo Research, Freiburg, Germany) according to the manufacturer's protocol. Following purification, library fragments were amplified by using the NEB Next® High-Fidelity 2X PCR Master Mix (New England Biolabs, Ipswich, UK) and custom-made indexed Nextera PCR primers (Sigma Aldrich, St. Louis, MO, USA). Libraries were amplified for a total of 15 cycles. SPRI select beads (Beckman Coulter GmbH, Krefeld, Germany) were used for library clean-up according to the manufacturer's protocol. CUT&Tag library preparation was carried out applying the protocol published by Kaya-Okur et al. (DOI: 10.1038/s41467-019-09982-5) Libraries were sequenced on Illumina NovaSeq XPlus platforms following the manufacturer's protocols.