cell line: HBG3 chip antibody: H3K4me3 time: Day2 agent: RA
Growth protocol
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 Day2+8hrs RA
cell line: HBG3 chip antibody: H3 time: Day2 agent: RA
Growth protocol
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 Day2+8hrs RA
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.
