title: Illumina HiSeq 2500 sequencing; Establishment of Porcine and Human Expanded Potential Stem Cells (ChIP-seq)
design: Establishment of Porcine and Human Expanded Potential Stem Cells (ChIP-seq)
library_construction_protocol: The C5 hiPSC was reprogrammed from the somatic cells derived from post-mortem human fetal neural tissue which was acquired at the time of surgical termination of pregnancy in the day surgery unit of Addenbrookes Hospital, Cambridge. Full informed consent was obtained from participating mothers. All generated lines are unlinked anonymous.. All ​related procedures and experiments were approved by the hospital and Sanger institute. M10 cells were derived from pig fetus fibroblast by reprogramming. Those pig fetus fibroblast cells were derived from a 28-day normal healthy male pig fetus skin. K3 cells were derived from 5-day preimplantation embryos, again, healthy male. All the related embryos and fetus experiments were approved by the Niedersaechsisches Landesamt fuer Verbraucherschutz und Lebensmittelsicherheit, LAVES, Oldenburg Germany. Abbreviations used: PFF means the pig fetus fibroblast. Emb means the origin is 5-day embryos. Parth means parthenogenetic embryos, PGCLC means the primordial germ cell-like cells differentiated from K3 cells.  Cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. 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. 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 Diagenode MicroPlex ChIP kit.
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-H3K4me3 (Diagenode C15410003-50)
attributes.tag: Experimental Factor: organism
attributes.value: Homo sapiens
attributes.tag: Experimental Factor: cell line
attributes.value: H1-hESC