CTCF ChIP-seq from mouse embryonic stem cell and their derivative neural stem cells
CTCF ChIP-seq from mouse embryonic stem cell and their derivative neural stem cells
Cells were detached with Accutase, counted and crosslinked with 1% (final) formaldehyde in their medium. Reaction was quenched with 0.125M (final) glycine, cell pellets were washed with PBS, snap frozen and stored in -80 freezer. Pellet of 10 million cells was used for CTCF ChIP experiment. ES (2i/LIF) cells were grown on gelatin-coated culture plastic in embryonic stem cells medium in the presence of MEK inhibitor (PD0325901) and GSK3 inhibition (CHIR99021) with LIF to maintain ESCs in a naive ground state. Cells were split at 80% confluence with a density of 30000 cells/sq.cm. The medium was exchanged daily.  NS cells were grown on laminin-coated plates in neural differentiation medium containing N2 and B27 supplements along with recombinant murine EGF and bFGF to a final concentration of 10 ng/ml. Cells were split at 80% confluence with a density of 35000 cells/sq.cm. The medium was exchanged daily.  CTCF depletion was carried out in CTCF-AID-GFP ES and NS Cells by treating with 500uM of indole acetic acid (IAA) dissolved in culture media for 24h Ddx5 depletion was carried out in Ddx5-FKBP NS Cells by treating with 500nM of dTAG13 dissolved in culture media for 24h ChIP prodecure was performed as described before (Pekowska et al., 2018, Cell). Libraries were constructed using Ovation Ultra System V2 (Tecan, 0344NB-32), according to manufacturer's instructions.