GSM8322916: S4KO_EB_Dnmt3b CUT&Tag_2; Mus musculus; OTHER
For ChIP-Seq sample preparation, ES or EB cells are treated with Activin or SB505124 as indicated, fixed and quenched, sonicated to average fragment size of 250bp in 1% SDS lysis buffer, and incubated with 60ul Dynabeads protein G conjugated with 3-5ug of indicated antibodies. 2% pre-cleared chromatin prior to primary antibody addition was kept as input DNA. Magnetic beads were washed, chromatin was eluted, and reverse crosslinked ChIP DNA was dissolved in 10 mM Tris pH 8.0 buffer for further analysis. For CUT&Tag, 1*10^5 ME cells or EB cells treated either with or without Activin as indicated, were harvested and crosslinked with 0.1% formaldehyde if necessary. Followed steps were performed using a commercially available kit (Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro, Vazyme, TD904). For ChIP-Seq library construction, ChIP-Seq DNA samples were quantified and quality assessed by Qubit and AATI Fragment Analyzer. DNA fragments range from 200-600bp were selected constructed for ChIP-Seq library with VAHTS® Universal Pro DNA Library Prep Kit for Illumina (Vazyme, ND608) according to manufacturer's instructions. For CUT&Tag library construction, purified DNA samples were carried out following manual (Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro, Vazyme, TD904)