The binding sites of SMAD2/3, Dnmt3a and Dnmt3b in pluripotent mESC, EBs and ME when Smad4 is lost with or without Dnmt3b knockdown

To understand the genome wide binding profile of SMAD2/3 when SMAD4 is lost, Smad2/3 ChIPseq was performed in SMAD4 knockout (S4KO) mouse embryonic stem cells (mESC) or in S4KO embryonic bodies (EBs). To figure out the effect of Dnmt3b on SMAD2/3 binding ability in the absence of SMAD4, we further performed SMAD2/3 CUT&Tag in EBs of S4KO and S4KO with Dnmt3b konckdown, as well as in mesendoderm (ME) stage of WT and S4KO. The binding sites of Dnmt3a and Dnmt3b were explored by Dnmt3a or Dnmt3b ChIP-seq and Dnmt3b CUT&Tag in WT EBs and S4KO EBs. WT with Dnmt3b knockdown and S4KO with Dnmt3b knockdown were used to as a control for the Dnmt3b CUT&Tag. Besides, we inserted a 3xFlag-tag at the C-terminus of Dnmt3a or Dnmt3b and then carried out Flag CUT&Tag in EBs. Overall design: For Smad2/3 ChIP-seq, S4KO cells were collected at ES (D0), embryoid body (EB) Day 3.5 treated with Activin (AC) or with SB505124(SB). For SMAD2/3 CUT&Tag, S4KO cells and S4KO with Dnmt3b-shRNA knockdown cells were harvested at EB Day 3.5 treated with AC. WT and S4KO were collected after mesendoderm (ME) induction for SMAD2/3 CUT&Tag. WT and S4KO cells on EB Day 3.5 after AC treatment were used for Dnmt3a or Dnmt3b ChIP-seq. Dnmt3b and Flag CUT&Tag were carried out in D3.5 EBs of WT, WT with Dnmt3b-shRNA knockdown, S4KO, S4KO with Dnmt3b-shRNA knockdown cells or Flag-tagged cells, respectively.