Illumina NovaSeq 6000 paired end sequencing; Simulated microgravity regulates the self-renewal and pluripotency of mouse and human embryonic stem cells - ChIP-seq
Simulated microgravity regulates the self-renewal and pluripotency of mouse and human embryonic stem cells - ChIP-seq
Cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. To stabilize Dpf2 on chromatin, cells were treated with 2 mM disuccinimidyl glutarate (DSG) for 10 minutes prior to formaldehyde crosslinking. For all other targets, cells were chemically cross-linked at room temperature by the addition of formaldehyde to 1% final concentration for 10 minutes and quenched with 0.125 M final concentration of glycine. Cross-linked cells were re-suspended in sonication buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS) and sonicated using a Diagenode Bioruptor for three 10-minute rounds using pulsing settings (30 sec ON; 1 min OFF). 10 µg of sonicated chromatin was then incubated overnight at 4ºC with 5 µg of Flag antibody conjugated to magnetic beads. Following the IP, beads were washed twice with RIPA buffer (50mM Tris-HCl pH8, 150 mM NaCl, 2mM EDTA, 1% NP-40, 0.1% Na-deocycholate, 0.1% SDS), low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10mM Tris pH 8.1, 250mM LiCl, 1mM EDTA, 1% Na-deoxycholate, 1% NP-40), and 1X TE. Finally, DNA was extracted by reverse crosslinking at 65ºC overnight with proteinase K (20ug/µL) and 1% SDS followed by phenol:chloroform:isoamyl alcohol purification and ethanol precipitation. Libraries were constructed as indicated above and sequenced using Illumina HiSeq 2500. Mouse E14 ESCs were cultured in DMEM (Hyclone) supplemented with 10% FCS (Gibco), 1xNEAA (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM L-glutamine (Gibco), and LIF (Millipore) on 0.1% gelatin (Sigma) coated plates. ChIPed DNA was purified using Qiagen MinElute PCR Purification Kit. Library DNA was purified using AmpureXP beads using 1:1 ratio. Libraries were constructed using the Illumina Nextera DNA Library Preparation Kit.