title: ES_AID_KI_CTCF-AID-GFP_IAA
organism: Mus musculus
description: Protocols: Cells were detached with Accutase, counted and 500,000 cells were lysed with TRI-Reagent (Sigma). 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 RNA was isolated using Direct-Zol RNA Isolation kit (ZYMO), according to the manufacturer's instructions. 1 ug of RNA (RIN > 8) was used for library constructions. Libraries were prepared using KAPA HyperPrep Kit, according to manufacturer's instructions. UDI/UMI (itDNA) indexes were used. Library quality was assessed with 4200 TapeStation System, High Sensitivity D1000 ScreenTape (Agilent).