Illumina HiSeq 2000 paired end sequencing; DNase-seq profile of mES cells treated with PFA/Noco/5aza, at different timepoints within in vitro mouse differentiation protocols, or in the presence of a NFYA dominant negative construct
Mouse embryonic stem cell culture and endoderm differentiation was modified slightly from previously published protocols (PMID: 15020474). Undifferentiated 129P2/OlaHsd mouse ES cells were maintained on gelatin-coated plates with mouse embryonic fibroblast (MEF) feeders in mES media composed of Knockout DMEM (Life Technologies) supplemented with 15% defined fetal bovine serum (FBS) (HyClone), 0.1mM nonessential amino acids (Life Technologies), Glutamax (Life Technologies), 0.55mM 2-mercaptoethanol (Sigma), and 1X ESGRO LIF (Millipore). Cells were regularly tested for mycoplasma and normal karyotype. Prior to differentiation, ES cells were passaged onto gelatin-coated plates for 25 minutes to deplete MEFs. MEF-depleted ES cells were then seeded at 1X10e4 cells/cm2 onto gelatin- coated dishes in mES media. After 12-24 hours, media was changed to Advanced DMEM (Life Technologies) supplemented with N-2 (Life Technologies), B27 Supplement without vitamin A (Life Technologies), and Glutamax. After 44-48 hours, media was changed to Advanced DMEM with 2% FBS, Glutamax, 5 nM GSK-3 inhibitor XV and 50 ng/mL E. coli-derived Activin A (Peprotech) for 24 hours to produce mesendoderm. For endoderm differentiation, cells were then fed with Advanced DMEM with 2% FBS, Glutamax, 50 ng/mL Activin A and 1 uM Dorsomorphin (Sigma) for 48 hours. For intestinal endoderm differentiation, cells at the endoderm stage were fed for 24 hours with Advanced DMEM with B-27 supplement without vitamin A, Glutamax, and 100 nM GSK-3 inhibitor XV. For pre-pancreatic endoderm differentiation, cells at the endoderm stage were fed for 24 hours with Advanced DMEM with B- 27 supplement without vitamin A, Glutamax, 500 nM retinoic acid (Calbiochem), 50 nM A-83-01 (Calbiochem), and 8 ng/mL Bmp4 (Stemgent). For mesodermal differentiation, cells at the mesendoderm stage were treated for 48 hours with 10 ng/mL Bmp4. ES cells with doxycycline-inducible alleles for Sox2, Foxa1, Hnf1beta, Cdx2, Gata6, Zfp161, and Klf7 in the HPRT locus were created as described (PMID: 22955985) and maintained and differentiated as above. For dominant negative lines, DNA-binding domains of NFYA and Nrf1 were used to create doxycycline-inducible HPRT lines as above. Dominant negative lines were grown for >7 days in mES media supplemented with 5 nM GSK-3 inhibitor XV and 500 nM UO126 to enhance pluripotency37 and 2 ug/mL Doxycycline. Cells were harvested at this stage for DNase-qPCR. For ChIP-qPCR, cells were treated for 6 hours with mES media with 1 uM retinoic acid. Cells were washed twice with 2.5 ml/plate of ice-cold PBS and pelleted (800g, 5 minutes, 4C). The supernatant was aspirated and cells were resuspended 2.5 mL/plate Buffer A containing 15 mM Tris HCl (pH 8), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (Ambion, pH 8), 0.5 mM EGTA (pH 8) and 0.5 mM Spermidine (Sigma-Aldrich). An equal volume of 2X Igepal in Buffer A (earlier samples with a 0.05% Igepal final concentration, later samples with a 0.00165% Igepal final concentration) was added to the resuspended cells and the nuclei were pelleted (800g, 5 minutes, 4C). The supernatant was aspirated promptly and the cells resuspended in 2.5 mL/plate Buffer A. An aliquot of the resuspended nuclei was taken and counted on a hematocytometer with 1:10 Trypan Blue. Nuclei were pelleted (800g, 5 minutes, 4C) and either digested immediately as below, or stored in 20 mM Tris HCl (pH 8), 75 mM NaCl, 0.5 mM EDTA (Ambion) 50% glycerol, 0.85 mM DTT, and 0.125 mM PMSF, flash frozen in liquid nitrogen and stored at -80C for a later digestion. If frozen, nuclei were washed twice with 2.5 mL/plate Buffer A, pelleted (800g, 5 minutes, 4C) and the supernatant discarded before proceeding to digestion. Digestion volumes are given for 10e7 nuclei. Volumes should be scaled as per cell number. Digestion buffer (850 ul per 107 nuclei) was prepared by diluting 10X stock of CaCl2 and NaCl in Buffer A (above) for a final concentration of 6 mM CaCl2 and 75 mM NaCl. Stop buffer (950 ul per 10e7 nuclei) was prepared with final concentrations as follows: 50 mM Tris HCl (pH 8), 100 mM Na Cl, 0.1 % SDS, 100 mM EDTA (Ambion, pH 8), 10 ug/ml RNase A (Invitrogen), 1 mM Spermidine (Sigma-Aldrich) and 0.5 M Spermine (Sigma-Aldrich). Both digestion and stop buffers were warmed at 37C. DNaseI enzyme was combined with warmed digestion buffer for a final volume of 100 ul per 107 nuclei. If multiple concentrations of DNaseI were tested, nuclei were separated equally amongst each concentration. Prior to digestion, a small aliquot was reserved as an undigested control for quantitative PCR analysis. The DNaseI/ digestion buffer mix was warmed in a 37C water bath for 2 minutes, and the nuclei were gently resuspended in digestion buffer. The warmed DNaseI/ digestion buffer mix was promptly added to the nuclei and digested for 2 minutes at 37C. Pre- warmed stop buffer was added and the tube inverted multiple times to stop the digestion. Digested samples were transferred to a 55C incubator for 15 minutes. Proteinase K (Ambion, 20 mg/mL) was added to each sample for a final concentration of 2ug/mL and samples were incubated overnight at 55C. DNA fragments were purified by adding an equal volume of phenol:chloroform:isoamyl alcohol to each sample and mixing. The suspension was transferred to a phase-lock tube and centrifuged for 5 minutes at 12-16,000g. The aqueous phase was isolated and combined with 0.1 volumes 3M sodium acetate, 2 volumes absolute ethanol and 1:600 Glycoblue. This mixture was incubated at -20C for 5-6 hours or at -80C for 30-45 minutes. DNA was pelleted by centrifugation at 12-16000g for 10-20 minutes and the supernatant aspirated. The DNA pellet was washed with 70% ethanol and re-pelleted by centrifugation at 4000 rpm for 5 minutes. The supernatant was carefully aspirated and the DNA pellet air-dried for 5-10 minutes before resuspending in TE buffer. To enrich for hypersensitive regions, size selection was performed on the purified fragments by either 2% agarose gel band purification or the Invitrogen E-Gel Agarose System. Fragments were collected in the 175-400 bp region so as to avoid empirically-determined complications by the 150 bp nucleosomal signal. Size selection enrichment was tested by quantitative PCR using primers (three positive control primers and three negative control primers) for constitutively DNase hypersensitive regions and insensitive regions as positive and negative controls respectively, on both the samples as well as the reserved undigested controls. Size selection enrichment was calculated as follows: Enrichment = 2^((Ct(negative control primer)-Ct(positive control primers))digested, size selected fragments-(Ct(negative control primer)-Ct(positive control primers))undigested control). DNA samples are confirmed using the Advanced Analytical Fragment Analyzer (FA) and prepared for sequencing using standard Illumina ligation based library construction on a SPRIworks system (Beckman Coulter Genomics). Ligated fragments are amplified for 8-16 cycles of PCR using standard Illumina primers containing molecular indexes. Final libraries are quantified on the FA and by qPCR before pooling and sequencing.