Illumina HiSeq 2500 paired end sequencing; Epigenetic regulation and transcriptional repression by Tet1 in the early post-implantation mouse embryo: ChIP seq
Epigenetic regulation and transcriptional repression by Tet1 in the early post-implantation mouse embryo: ChIP seq
Cells were collected from a wild-type ES cell line electroporated with pPyCAGIP vector expressing JMJD8(27-271)-V5/His  homozygous cells for Tet1 gene trap GT(RRG140)  were collected from B6 incipient congenic (N5; >95% B6) blastocyst-derived embryonic stem cell line (KO) converted in vitro to epiblast-like cells. Cells were trypsinized and fixed in freshly prepared 1% para-formaldehyde at room temperature for 10 min and quenched with 1.25M glycine. Nuclei were isolated and chromatin was sheared to 200-500bp using Bioruptor sonicator for 20 cycles (30s ON, 30s OFF). Sheared chromatin was immunoprecipated using appropriate antibody and incubated overnight with Dyna beads Protein G (10004D). Immunocomplexes were washed for 5 minutes each with the following buffers: 1X low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), 1X high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), 1X LiCl buffer (10mM Tris pH 8.1, 250mM LiCl,1mM EDTA, 1% deoxycholate, 1% NP-40) and 2X TE buffer (+ 50mM NaCl). Chromain was eluted with elution buffer (50 mM Tris-Hcl, pH 8.0, 10 mM EDTA and 1% SDS) and reverse cross-linked by incubation at 65 °C in 5M NaCl buffer over night. DNA was purified using Zymo ChIP DNA Clean & Concentrator Kit (D5205) for library preparation. Library was prepared using NEBNext Ultra DNA Library Prep Kit and sequenced on an Illumina HiSeq 2500
Experimental Factor: IgG (SantaCruz sc-2027)
immunoprecipitate
Experimental Factor: B6 incipient congenic (N5; >95% B6) epiblast-like cells
cell type
Experimental Factor: wild type
genotype