title: GSM7883391: cr_myod_dtag43 (matr3.ko and yy1.wt) (for double ko comparison); Mus musculus; OTHER
library_construction_protocol: CUT&RUN experiments were carried out as described (Janssens et al., 2019, Meers et al, 2019) with modifications. Briefly, ~0.5x106 cells were collected and incubated with primary antibody on a nutator at 4°C overnight. After washing away unbound antibody, protein A-MNase was added at 700ng/ml and incubated for 1h at 4°C. Samples were washed again and placed in a 0°C metal block. To activate protein A-MNase, CaCl2 was added to a final concentration of 2mM. The reaction was incubated at 0°C for and stopped by chelating Ca2+. The CUT&RUN fragments were released from the insoluble nuclear chromatin at 37°C for 30min. Digested chromatin in the supernatant were collected and then digested by proteinase K at 50°C for 1h. DNA was extracted by phenol chloroform extraction, ethanol precipitation. The antibodies used were: Matr3, Proteintech, 12202-2-AP; CTCF, Abcam, ab70303; Rad21, Abcam, ab992; YY1, santa cruz, sc-7341X; MyoD, Santa cruz, sc-32758 X; H3K27Ac, millipore, MABE647; H3K4me3, active motif, 39159; Rabbit Anti-Mouse IgG H&L, abcam, ab46540. For transcription factors (TF), libraries were constructed using the library preparation manual of “NEBNext® Ultra™ II DNA Library Prep Kit for Illumina”, NEB E7645, with modification (Liu et al., 2018). Briefly, dA-tailing temperature was decreased to 50°C to avoid DNA melting, and the reaction time was increased to 1 hour to compensate for lower enzymatic activity. After adaptor ligation, 1.87x volume of AMPure XP beads was added to the reaction to ensure high recovery efficiency of short fragments. After 12 cycles of PCR amplification, the reaction was cleaned up with 1.2x volume of AMPure XP beads. For histone markers, libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina”, NEB E7645, per manufacturer's instruction with modification. Briefly, after end repair and adapter ligation, 1.1x volume of AMPure XP beads was added to the reaction for DNA cleanup. After 12 cycles of PCR amplification, the reaction was cleaned up with 1.1x volume of AMPure XP beads. 16-24 barcoded libraries were quantified and mixed at equal molar ratio. Library was loaded to a NextSeq 500/550 High Output Kit v2 (75 cycles), and sequenced in the NextSeq 500 platform. To enable determination of fragment length, paired-end sequencing was performed (2x42 bp, 6 bp index)