Cell Culture
Human fetal skeletal myoblasts (a generous gift from Dr. Eric Shoubridge) were cultured in SKGM-2 media (Cat. # cc-3245, Cambrex, East Rutherford, NJ) containing 10% fetal bovine serum (Hyclone, Logan, Utah) 10 ng/ml human epidermal growth factor, 0.1 mg/mL insulin, 0.5 mg/mL BSA, 0.5 mg/mL fetuin, 0.39 μg/mL dexamethasone and 50 μg/mL gentamicin and maintained at 37°C and 5% CO2. Cells were grown to approximately 70% confluence and were induced to differentiate in to multinucleated myotubes by mitogen withdrawal. Cells were maintained in differentiation media (DMEM supplemented with 2.5% horse serum) for five days prior to harvesting.
RNA extraction and hybridization
Total RNA was extracted from 1 × 106 myoblasts and differentiated myotubes using the RNeasy Mini Kit (Qiagen, Valencia, CA (5 μg) according to the manufacturer's instructions. RNA was quantified, quality-checked by the Bioanalyzer (Agilent) and reverse-transcribed with a cDNA synthesis kit in the presence of SuperScript II RT (Invitrogen-Life Technologies, Inc.) and an oligo dT-T7 primer (Affymetrix Inc., Santa Clara, CA). Ten microliters of purified cDNA were used for the in vitro transcription (IVT) amplification reaction, in the presence of biotinylated nucleotides (Enzo Biochem Inc.). Labeled cRNA (15 μg) was fragmented by incubation at 94°C for 35 min in fragmentation buffer (GeneChip Sample Cleanup, Qiagen) and hybridized competitively against the Affymetrix HG-U133 microarray set. Arrays were scanned using a GeneArray 2500 scanner (Affymetrix) and analyzed using MicroArray Suite 5.0 (Affymetrix).
DNA extraction and chromatin immunoprecipitation
Total genomic DNA was extracted from myoblasts using the Qiagen DNeasy Tissue Kit (Valencia CA) and was used as a control for the SNP-Array studies. Antibody used in Chromatin immunoprecipitation was anti-acetyl-Histone H4 peptide corresponding to amino acids 2–19 of Tetrahymena histone H4 acetylated on Lys5, Lys8, Lys12 and Lys16 (Upstate Cat #06–866). Chromatin immunoprecipitation was performed using the ChIP Assay kit from Upstate Biotech (Charlottesville, VA) (Cat# 17–295) as per manufacturer's instructions. Briefly, 1 × 108 cells were cross-linked for 15 minutes at room temperature with 1% formaldehyde. Cells were washed twice with ice-cold PBS, scraped from tissue culture plates and subsequently resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.1) supplemented with protease inhibitors (Mini Complete, Roche, Cat. # 1836153) and lysed for 10 minutes on ice. DNA was sheared to between 1 and 2 kb by sonication for three 15-second pulses with a 1.5 mm step probe equipped sonicator set to a magnitude of 30%. Sonicated lysates were cleared by centrifugation for 10 minutes at 13,000 rpm at 4°C. Cleared lysates were diluted 10 fold in 0.01% SDS, 1.1% Triton ×-100, 1.2 mM EDTA, 16.7 mM Tris HCl, pH8.1, 167 mM NaCl and incubated with 70 μl of Salmon Sperm DNA/Protein A Agarose-50% slurry (Upstate Biotech, Cat# 16–157C) to reduce non-specific background for 1 hour at 4°C with rotation. Five μls of Anti-acetyl-H4 (Upstate Biotech, Cat. # 06–866) was added to the lysates (per 106 cell equivalents). Immune complexes were recovered with Salmon Sperm DNA/Protein A Agarose and washed twice with low salt buffer (0.1% SDS, 1% Triton ×-100, 2 mM EDTA, 20 mM Tris HCl, pH8.1, 150 mM NaCl); twice with high salt buffer (0.1% SDS, 1% Triton ×-100, 2 mM EDTA, 20 mM Tris HCl, pH 8.1, 500 mM NaCl); twice with LiCl buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris HCl, pH 8.1), and twice with TE. Immune complexes were eluted with an elution buffer containing 1% SDS and 0.1 M NaHCO3 for 30 min at room temperature with rotation. DNA-protein crosslinks were reversed for 4 hrs at 65°C with 0.2 mM NaCl. Samples were deproteinated and DNA isolated using columns as described by the manufacturer (Qiagen). DNA was eluted from columns with 30 μls of ddH2O.
Target Preparation
Immunoprecipitated DNA and control (whole-cell extract) DNA were assayed according to the protocol (GeneChip Mapping Assay manual) supplied by Affymetrix. Briefly, a total of 250 ng DNA was digested with XbaI then ligated to XbaI adaptor before PCR amplification. Cycling conditions were: 95°C for 3 minutes followed by 35 cycles of 95°C for 20 seconds, 59°C for 15 seconds, and 72°C for 15 seconds. Final extension was done at 72°C for 7 minutes (DNA Engine Tetrad PTC-225, MJ Research, Waltham, MA). To evaluate PCR products, 3 μL of each PCR product was mixed with 3 μL of 2× gel loading dye on 2% Tris-borate EDTA gel and run at 120 V for 1 hour to check for the expected product between 250 and 1,000 bp. Twenty μg of PCR product was fragmented with DNase I and biotinylated overnight at 37°C using biotin-N6-ddATP (Perkin Elmer) and terminal transferase (Promega, Madison WI). Target hybridization, washing, scanning and staining were performed as recommended by the manufacturer.
Genotype generation
The 10 K SNP arrays were scanned with the Affymetrix GeneChip Scanner 3000 using GeneChip Operating System 1.0 (Affymetrix). Data files were generated automatically and genotype calls were made automatically by GeneChip DNA Analysis Software 2.0 (Affymetrix). Genomic location of SNP probesets was derived using the Affymetrix GeneChip Mapping 10 K library file (Mapping10K_Xba131) and the Ensembl human genome map (v27.35a.1), based on the release 35 of the human genome sequence (May 2004). Each probeset is identified in the Affymetrix annotation by its tscID (The SNP Consortium). These IDs are mapped to rsIDs (NCBI RefSNP ID), which are used to identify SNP positions in the Ensembl database.
SNP-Array probeset selection and analysis
This study used a pre-commercial release version of the Affymetrix GeneChip Mapping 10 K SNP Array for the human genome (ax13339). This microarray contains probesets for 10,043 SNPs distributed across the 22 autosomes and the X chromosome. An initial selection process identified probesets that map to an unambiguous location in the human genome; tscIDs were mapped to rsIDs, for which genomic locations can be found in Ensembl. Screening removed 448 tscIDs, which could not be mapped to any rsID; 2,362 rsIDs not localized in Ensembl; and 33 with multiple locations. The remaining 7,200 probesets, which have a unique location, were used in our analysis.
As a further selection for high quality SNP data, we performed a control genotyping experiment in triplicate using the total DNA from the same myoblast cell line. Of the 7,200 uniquely mapped probesets, only the 6,464 for which the same genotyping was obtained in at least two of the three replicates were used for our analysis.
The analysis of SNP-array data was performed in biological triplicates. We analyzed the results from SNP arrays using the Genotyping Tools V 1.0 (Affymetrix). This tool assesses whether measurable hybridization to a probeset is present, and provides heterozygosity data. Probes were classified as hybridizing or not based on calls of Present (P) (present in at least two of three replicates) or Absent (A) (absent in at least two of three replicates). All other probesets were not included in further analysis.
DNA microarray probeset selection and analysis
The Affymetrix HGU133A/B gene expression array set contains 44,760 probesets from genes on the 22 autosomes and on chromosomes X and Y. We selected the probesets that were unambiguously mapped to a genomic position in the NetAffx probeset annotations [17] (April 12, 2005, http://www.affymetrix.com/, based on NCBI release 35 of the human genome sequence). Screening removed 858 probesets without a genomic location in NetAffx, 3,114 with multiple locations, and 52 corresponding to genes in chromosome Y, which is not covered by the SNP Array. The remaining 40,736 probesets with a unique genomic location were used in our analysis.
We analyzed the results from the expression arrays using the MicroArray Suite 5.0, (Affymetrix). This tool assesses whether measurable hybridization to a probeset is present. Analysis of gene expression was performed in biological triplicates. Probesets were classified as hybridizing or not based on calls of Present (P) (present in at least two of three replicates) or Absent (A) (absent in at least two of three replicates), respectively. All other probesets were not included in further analysis.
Availability of microarray data in GEO
We have deposited all microarray data used in this work at the Gene Expression Omnibus database (National Center for Biotechnology Information) where it is available under the super-series identifier GSE4133. This super series is composed of the following subset series: GSE4131 (triplicate Affymetrix HGU133A/B expression chips hybridized to RNA extracted from myoblasts and myotubes) and GSE4132 (triplicate Affymetrix 10 K ax 13339 SNP-chips were hybridized to DNA from myoblasts (control), myoblast ChIP, and myotube ChIP). The gene expression data is also available at the StemBase database of stem cell gene expression data [18] under experiment identifier E204 (containing sample S267 for the myoblasts, and sample S273 for the myotubes).
