library_construction_protocol: ChIP DNA was extracted by Native ChIP protocol The ChIP DNA was fragmented by Picoruptor (Diagenode) for 10 cycles of 30 seconds on, 30 seconds off. Then, ChIP library was constructed by KAPA Hyper Prep Kit (KAPA BIOSYSTEMS) and SeqCap Adapter Kit A (Roche) according to manufacturer instructions. The concentration of the ChIP-seq library was quantified by KAPA Library quantification kit (KAPA BIOSYSTEMS). ChIP sequencing was performed on a HiSeq X platform (Illumina). H2Aub CATCH-seq libraries were prepared as previously described (Zhu et al., 2021) with some modifications. Briefly, 2,000 MEFs per sample were used for library construction. Cells were permeabilized by Nuclei EZ lysis buffer supplemented with 0.1% Triton, 0.1% deoxycholate, complete EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride on ice. For each sample, 800 S2 Drosophila culture cells were added for a spike-in normalization purpose. Chromatin was fragmented in situ by 2 U/µl MNase (M0247S, NEB) at 37C for 7.5 min. The reaction was stopped by adding 1/10 volume of 100 mM EDTA and diluted in the freshly prepared immunoprecipitation buffer. As an input, 5% of the chromatin lysate was taken. Then, 30 ng of annealed I-SceI carrier DNA was added to each sample. The forward and reverse strands of the carrier DNA were as follows: /5AmMC6/Gtagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggataacagggtaattagggat aacagggtaat*c/3AmMO/ and /5AmMC6/Gattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatccctaattaccctgttatcc cta*c/3AmMO/, respectively, where asterisks represent phosphorothioate bonds. The oligos were synthesized by Integrated DNA Technologies. For each immunoprecipitation reaction, 0.5 µl of rabbit monoclonal anti-H2AK119 monoubiquitylation antibody (Cell Signaling, #8240) conjugated to precleared Dynabeads Protein A and G mixture was used. After immunoprecipitation at 4C overnight, the chromatin-Dynabeads were washed by the low and high salt wash buffers, and the chromatin was eluted in the freshly prepared ChIP elution buffer at 65C for 1 hr. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. Adaptor ligation was pre-formed by NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB) in a half scale of the manufacturer's instruction, and the libraries were purified by 1.8x SPRIselect beads (B23318, Beckman Coulter). The DNA was amplified by KAPA Hifi 2X mater PCR mix (KK2605) for 14 PCR cycles with indexing primers. After purification with 0.9x SPRIselect beads, the samples were digested by I-SceI (5 U/µl, NEB, R0694) 37C for 2 hrs followed by heat inactivation at 65C for 20 min and purified by 0.9x SPRIselect beads. The second amplification was not performed. The libraries were sequenced on a HiSeq X.