title: GSM2417158: 48h_Sox2_SKpMX_ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: Histone Modifications were assesed by means of Native ChIP. 50 x 106 Nuclei were isolated from non-crosslinked cells (MEFs, 48hOSKM, pre-i#1 and ESC) by incubation in 2 mL of a hypotonic solution (0.3M sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl2, 15mM Tris-HCl pH 7.5, 0.5mM DTT, 0.1% NP40, and protease inhibitor cocktail) followed by subsequent centrifugation through a sucrose cushion (1.2M sucrose, 60mM KCL, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 15 mM Tris-HCl pH 7.5, 0.5mM DTT, and protease inhibitor cocktail). Nuclei were then re-suspended in MNAse-digestion buffer (0.32M sucrose, 50mM Tris-HCl pH 7.5, 4mM MgCl2, 1mM CaCl2, and protease inhibitor cocktail) and digested with 3 units of MNase (Roche 10107921001) for 10 minutes at 37oC. The first soluble fraction (S1) was recovered by centrifugation for 10 min at 10,000 rpm. The nuclei pellet was then dialyzed overnight in 1l of dialysis buffer (1mM Tris-HCl pH7.5, 0.2mM EDTA, protease inhibitors) to more completely release the chromatin fraction (S2) from nuclei. 10 ug of soluble chromatin (S1 and S2) was then incubated with 5 ug of antibodies targeting histone modifications-conjugated to magnetic beads (Active Motif; 53014) under constant stirring at 4oC for 16 hrs. The antibodies used were anti-H3K9ac (Abcam; ab4441), anti-H3K4me3 (Abcam; ab8580), anti-H3K4me2 (Abcam ab7766), anti-H3K4me1 (Abcam; ab8895), anti-H3K27me3 (Active Motif; 39155), antiH3K27ac (Abcam; ab4729), and anti-H3K36me3 (Abcam; ab9050). Beads were washed twice with wash buffer A (50mM Tris-HCl pH 7.5, 10mM EDTA, 75mM NaCl), wash buffer B (50mM Tris-HCl pH 7.5, 10mM EDTA, 125mM NaCl), and wash buffer C (50mM Tris-HCl pH 7.5, 10mM EDTA, 175 mM NaCl). DNA was extracted using phenol:chloroform:iso-amylacohol and used for downstream library construction. DNA from fractions S1 and S2 was also isolated directly using phenol:chloroform:iso-amylacohol extraction and used as an whole genome input control (native Input). All protocols for Illumina/Solexa sequencing library preparation, sequencing, and quality control were performed as recommended by Illumina, with the minor modification of limiting the PCR amplification step to 10 cycles. All constructed libraries were sequenced using single-end 50 bp sequencing reactions. Transcription factor occupancy data generated in this study were acquired using ChIP after crosslinking cells. X-ChIP was also employed for mapping H3K79me2 (Active Motif, 39143), H3K9me3 (abcam, ab8898 or Millipore, 05-1242), H3 (abcam, ab1791) and H3.3 (Abnova, H00003021-M01). Briefly, cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. To stabilize HATs/HDACs (p300, Hdac1) and Brg1 on chromatin, cells were treated with 2 mM disuccinimidyl glutarate (DSG) for 10 minutes prior to formaldehyde crosslinking. For all other antibodies used, 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 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 ug of sonicated chromatin was then incubated overnight at 4oC with 5 ug of antibodies-conjugated to magnetic beads. The antibodies used were: anti-Esrrb (RnD; H6705), anti-Klf4 (RnD; AF3158), anti-cMyc (RnD; AF3696), anti-Nanog (cosmobio REC-RCAB001P), anti-Oct4 (RnD; AF1759), anti-Sox2 (RnD AF2018), anti-p300 (SantaCruz;sc-585), anti-Runx1 (Novus Biologicals NBP1-61277), anti-Fra1 (SantaCruz;sc-183X), anti-Cebpa (SantaCruz; sc-61X), anti-Cebpb (SantaCruz;sc-150X), anti-Hdac1(abcam; ab7028) and anti-Brg1(abcam; ab110641). Following the IP the 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 1xTE. Finally, DNA was extracted by reverse crosslinking at 60oC overnight with proteinase K (20ug/ul) and 1% SDS followed by phenol:chloroform:iso-amylacohol purification. Libraries were constructed as indicated above and sequenced using single-end 50 bp sequencing reactions Libraries were prepared according to Illumina's instructions accompanying the TruSeq library preparation kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM2417158