Doxycycline (dox)-inducible secondary iPSCs containing GFP targeted to the endogenous Nanog locus and constitutive nuclear RFP
time
48h
treatment
NoDox
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Cells were crosslinked in 1% formaldehyde for 10 minutes at 37°C with constant stirring, followed by quenching with 125 mM glycine for 5 min at room temperature. Cells were rinsed with 1X PBS and lysed, and chromatin was sheared using a Branson sonicator until the majority of DNA was in the range of 200-1,000 base pairs. Chromatin was incubated with antibody at 4°C overnight with constant stirring. Co-immunoprecipitation of antibody-protein complexes was performed using Protein A or Protein G Dynabeads for 1h at 4°C. ChIPs were completed using previously reported methods with the following antibodies: OCT4 (Santa Cruz, sc-8628x), H3K4me2 (Millipore, 07-030),H3K27ac () .Immunoprecipitated DNA was end repaired using the End-IT DNA End-Repair Kit (Epicentre), extended using Klenow fragment (3'-5' exo) (NEB), and ligated to sequencing adapter oligos (Illumina). Each library was PCR-amplified using PFU Ultra II Hotstart Master Mix (Agilent), and a size range of 300-600 base pairs selected for sequencing. Libraries were sequenced on the Illumina Hiseq 2000.