cell line: HeLa S3 cells (ATCC/National Cell Culture Center) antibody: anti-lamin B antibody, EMD Biosciences, NA12
Growth protocol
HeLa S3 cells were grown in Minimum Essential Medium supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 6 x10^5 cells/ml.
Extracted molecule
genomic DNA
Extraction protocol
HeLa S3 cells were fixed with 1% formaldehyde at room temperature for 10 min and the fixation was terminated by the addition of glycine to a final concentration of 125 mM. The fixed cells were washed in cold 1x Dulbecco’s PBS (Invitrogen) and swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 3 x10^8 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7x 30 s intervals) to an average size of less than 500 bp as verified on a 2% agarose gel. Lysates were then clarified by centrifugation for 15 min at 20,000 xg at 4°C. Chromatin immunoprecipitations from 1x10^8 cells were conducted as previously described (Euskirchen et al. 2007). Antibodies used were anti-lamin A/C (H-110), Santa Cruz Biotechnology, sc-20681, anti-lamin B antibody, EMD Biosciences, NA12 or normal IgG.
Label
Cy5
Label protocol
ChIP DNA prepared from 1x10^8 cells was directly labeled by Klenow (New England Biolabs) by random priming with Cy dye-coupled nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
cell line: HeLa S3 cells (ATCC/National Cell Culture Center) antibody: normal mouse IgG
Growth protocol
HeLa S3 cells were grown in Minimum Essential Medium supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 6 x10^5 cells/ml.
Extracted molecule
genomic DNA
Extraction protocol
HeLa S3 cells were fixed with 1% formaldehyde at room temperature for 10 min and the fixation was terminated by the addition of glycine to a final concentration of 125 mM. The fixed cells were washed in cold 1x Dulbecco’s PBS (Invitrogen) and swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 3 x10^8 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7x 30 s intervals) to an average size of less than 500 bp as verified on a 2% agarose gel. Lysates were then clarified by centrifugation for 15 min at 20,000 xg at 4°C. Chromatin immunoprecipitations from 1x10^8 cells were conducted as previously described (Euskirchen et al. 2007). Antibodies used were anti-lamin A/C (H-110), Santa Cruz Biotechnology, sc-20681, anti-lamin B antibody, EMD Biosciences, NA12 or normal IgG.
Label
Cy3
Label protocol
ChIP DNA prepared from 1x10^8 cells was directly labeled by Klenow (New England Biolabs) by random priming with Cy dye-coupled nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Hybridization protocol
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine and 0.1 ug/ul herring sperm DNA. Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html).
Scan protocol
Arrays were scanned on an Axon 4000B scanner.
Data processing
For each pair of arrays the files (in .GFF file format) corresponding to the two channels for ChIP DNA (635 nm) and reference DNA (532 nm), were uploaded to the TileScope pipeline for normalization and scoring (Zhang et al. 2007 Genome Biology, 8:R81). Three biological replicates were used per factor, and each biological replicate was hybridized separately to one array. Data were scored with the following Tilescope program parameters: quantile normalization of replicates, iterative peak identification, window size= 500, oligo length=50, pseudomedian threshold=1.0, p-value threshold=4.0, peak interval=1000, and feature length=1000. Regions called by Tilescope were then filtered and corrected for multiple hypothesis testing by false discovery rate (FDR). To generate our set of background regions for FDR analysis, we randomly shuffle the probe values within each replicate, ensuring that the same probes are swapped for each replicate. This shuffled data set is then used as input to Tilescope and the scores compared against the lamin A/C and the lamin B data sets. The final lists of enriched regions for lamin A/C and lamin B have a final FDR of 0.1. Target coordinates were converted to hg18 using the UCSC ‘liftOver’ utility (http://genome.ucsc.edu/cgi-bin/hgLiftOver) to yield laminAC-peaks.bed and laminB-peaks.bed.
