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. ESCs dissociated with 0.05% of trypsin were centrifuged and re-suspended in ES medium without LIF, and then plated in 25 ml cell culture flask to induce the formation of EBs. Cultures were maintained for indicated days and incubated at 37 °C with 5% CO2. EBs at indicated time were collected for qPCR analysis.A Rotary Cell Culture System (RCCS) (SM-31, National Space Science Center, Chinese Academy of Sciences, China) was used in this study to simulate microgravity. 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.