Novaseq 150pe

CUT&Tag is operated according to the manufacturer’s instructions for the Hyperactive Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme Biotech). In brief, WT and HBO1-KO HeLa cells were collected and counted for incubation with pre-treated ConA beads. Subsequently, cells are resuspended in an Antibody Buffer and incubated overnight at 4 °C with the corresponding primary antibody of HBO1 or H3K9la. The secondary antibodies were diluted in appropriate proportions and incubate them with the cells at room temperature. Subsequently, pA/G-Tnp transposons were rotated and incubated with the samples for 1 h to activate the translocase fragment DNA and extract DNA. DNA library was constructed with TruePreP Index Kit V2 for Illumina (TD202, Vazyme Biotech). The library was purified by VAHTS DNA Clean Beads (N411, Vazyme Biotech) and sequenced by Illumina novaseq 150PE.

For CUT&Tag, the Hyperactive In Situ ChIP Library Prep Kit for Illumina (pG‐Tn5) was utilized in compliance with the manufacturer's guidelines. Briefly, TBD0220 and TBD0220TR cells were bound using concanavalin A‐coated beads. After being resuspended in an antibody buffer, the cells underwent sequential treatment with primary and secondary antibodies directed against H3K9la. The samples and pA‐Tn5 transposase were incubated simultaneously. DNA was isolated, amplified, and purified after transposon activation and tagmentation in order to construct the library. Using VAHTS DNA Clean Beads, purification processes were completed following the construction of the sequencing library. The VAHTS Library Quantification Kit for Illumina was used to quantify the library before being sequenced on an Illumina Nova seq 150PE.

CUT&Tag was performed with a Hyperactive Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme Biotech) according to the manufacturer’s recommended protocol [25 (link)]. In brief, NSCs stably expressing Mysm1-Flag and induced astrocytes were collected to extract nuclei in NE buffer and then combined with ConA Beads. Subsequently, cells were resuspended in antibody buffer and incubated with primary antibodies against Flag (F1804, Sigma‒Aldrich) and secondary antibodies (Ab206-01, Vazyme Biotech). The samples were incubated with pA/G-Tnp transposase. After transposon activation and tagmentation, DNA was isolated, amplified, and purified to construct a library. The library for sequencing was constructed, and VAHTS DNA Clean Beads (N411, Vazyme Biotech) were used for purification steps. The library was quantified with a VAHTS Library Quantification Kit for Illumina (Vazyme Biotech) and sequenced on an Illumina NovaSeq 150PE. All cells were tested for mycoplasma contamination before experiment.