ES cells were grown on ES Medium (DMEM, 15% FBS, Non-essential amino acids, Glutamine, Pen/Strept, 2-mercaptoethanol and LIF) on feeder cells for amplification or on Gelatin for differentiation and experiments. Motor neuron differentiation was done as described (Wichterle & Peljto, Curr Protoc Stem Cell Biol, 2009). Briefly, ES cells were tripsinized and allowed to form aggregates in ANDFK medium (Advanced DMEM/F12, Neurobasal Medium, Knockout-SR, of Pen/Strep, L-Glutamine, and 2-mercaptoethanol. Day 0 is the day of trypsinization. At Day 2, RA and Hh agonist are added.
Extracted molecule
genomic DNA
Extraction protocol
ChIP protocols were adapted from http://jura.wi.mit.edu/young_public/hESregulation/ChIP.html. Briefly, approximately 3x10e7 cells taken from each developmental time point were cross-linked using formaldehyde and snap-frozen with liquid nitrogen. Cells were thawed on ice, resuspended in 5ml lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and mixed on a rotating platform at 4°C for 5 minutes. Samples were spun down for 3 minutes at 3000rpm, resuspended in 5ml lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and mixed on a rotating platform for 5 minutes at room temperature. Samples were spun down once more, resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp. Triton X-100 was then added to the lysate after sonication to final concentrations of 1% and the lysate spun down to pellet cell debris. The resulting whole-cell extract supernatant was incubated on a rotating mixer overnight at 4°C with 100 μL of Dynal Protein G magnetic beads that had been preincubated the previous 24 hours with 10 μg of the appropriate antibody in a PBS/BSA solution. After approximately 16 hours of bead-lysate incubation, beads were collected with a Dynal magnet. ChIP samples probing for transcription factor binding were washed with the following regimen, mixing on a rotating mixer at 4°C for 5 minutes per buffer: low-salt buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high-salt buffer (20 mM Tris at pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris at pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% deoxycholate, 1% NP-40), and TE containing 50 mM NaCl. ChIP samples probing for histone and chromatin marks were washed 4 times with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and then once with TE containing 50 mM NaCl, again mixing on a rotating mixer at 4°C for 5 minutes per buffer. After the final bead wash, samples were spun down to collect and discard excess wash solution, and bound antibody-protein-DNA fragment complexes were eluted from the beads by incubation in elution buffer at 65°C with occasional vortexing. Cross-links were reversed by overnight incubation at 65°C. Samples were digested with RNase A and Proteinase K to remove proteins and contaminating nucleic acids, and the DNA fragments precipitated with cold EtOH. Resulting purified DNA fragments were amplified via ligation-mediated (LM) PCR.
Label
Cy5
Label protocol
Amplified DNA was labeled with a BioPrime CGH Genomic Labeling System (Invitrogen, 18095-011)
Channel 2
Source name
H3 ChIP DNA from ES to motor neuron differentiation Day0
ES cells were grown on ES Medium (DMEM, 15% FBS, Non-essential amino acids, Glutamine, Pen/Strept, 2-mercaptoethanol and LIF) on feeder cells for amplification or on Gelatin for differentiation and experiments. Motor neuron differentiation was done as described (Wichterle & Peljto, Curr Protoc Stem Cell Biol, 2009). Briefly, ES cells were tripsinized and allowed to form aggregates in ANDFK medium (Advanced DMEM/F12, Neurobasal Medium, Knockout-SR, of Pen/Strep, L-Glutamine, and 2-mercaptoethanol. Day 0 is the day of trypsinization. At Day 2, RA and Hh agonist are added.
Extracted molecule
genomic DNA
Extraction protocol
ChIP protocols were adapted from http://jura.wi.mit.edu/young_public/hESregulation/ChIP.html. Briefly, approximately 3x10e7 cells taken from each developmental time point were cross-linked using formaldehyde and snap-frozen with liquid nitrogen. Cells were thawed on ice, resuspended in 5ml lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and mixed on a rotating platform at 4°C for 5 minutes. Samples were spun down for 3 minutes at 3000rpm, resuspended in 5ml lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and mixed on a rotating platform for 5 minutes at room temperature. Samples were spun down once more, resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp. Triton X-100 was then added to the lysate after sonication to final concentrations of 1% and the lysate spun down to pellet cell debris. The resulting whole-cell extract supernatant was incubated on a rotating mixer overnight at 4°C with 100 μL of Dynal Protein G magnetic beads that had been preincubated the previous 24 hours with 10 μg of the appropriate antibody in a PBS/BSA solution. After approximately 16 hours of bead-lysate incubation, beads were collected with a Dynal magnet. ChIP samples probing for transcription factor binding were washed with the following regimen, mixing on a rotating mixer at 4°C for 5 minutes per buffer: low-salt buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high-salt buffer (20 mM Tris at pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris at pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% deoxycholate, 1% NP-40), and TE containing 50 mM NaCl. ChIP samples probing for histone and chromatin marks were washed 4 times with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and then once with TE containing 50 mM NaCl, again mixing on a rotating mixer at 4°C for 5 minutes per buffer. After the final bead wash, samples were spun down to collect and discard excess wash solution, and bound antibody-protein-DNA fragment complexes were eluted from the beads by incubation in elution buffer at 65°C with occasional vortexing. Cross-links were reversed by overnight incubation at 65°C. Samples were digested with RNase A and Proteinase K to remove proteins and contaminating nucleic acids, and the DNA fragments precipitated with cold EtOH. Resulting purified DNA fragments were amplified via ligation-mediated (LM) PCR.
Label
Cy3
Label protocol
Amplified DNA was labeled with a BioPrime CGH Genomic Labeling System (Invitrogen, 18095-011)
Hybridization protocol
Labeled DNA samples were co-hybridized to custom Agilent DNA microarrays using the Oligo aCGH/ChIP-on-chip Hybridization Kit (Agilent, 5188-5220) at 65°C for approximately 16 hours. Arrays were washed according to previously published protocols (http://jura.wi.mit.edu/young_public/hESregulation/ChIP.html).
Scan protocol
Arrays were scanned at dual PMT intensities (10% and 100%) at a 5um resolution using an Agilent microarray scanner. Data from the two array scans were combined and extracted using the Agilent Feature Extraction software.
Description
H3K4me3 ChIP-chip timeseries during ES to motor neuron differentiation Day0
Data processing
Feature extraction was performed with Agilent Feature Extraction software. Background subtracted values were normalized with (1) median normalization, (2) line fitting normalization, (3) quantile normalization; all normalization code was implemented in SQL as part of our in-house microarray database. Median normalization accounts for differences in the amount of dye hybridized between the two channels. This normalization multiplied IP intensities by median(control)/median(IP) such that the median intensity of the two channels is the same. Line fitting normalization is similar to Loess normalization but with a simpler model; it assumes that the bulk of the data should fall along the line y=x. Line fitting performed linear regression on the IP values as a function of the control and then rotated the datapoints such that the resulting line had slope one and intercept zero; this transformation is performed in log space. Finally, in order to allow comparison of probe intensities across time-points, arrays were quantile normalized.
