M87A medium supplemented with oxytocin and cholera toxin at 0.5 ng/ml
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). 5 μg of RNase treated DNA was dried under vacuum and resuspended with 50 μl SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and 450 μl of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl). Vortex for ~20 minutes to fully resuspend the DNA. Sonicate the samples 5x for 10 seconds (5 times total) at 8 watts. Make sure that most of the DNA is between 300-600 bp long. Set aside 50 μl of sample in a new microfuge tube - Input DNA. [immunoprecipitation protocol] Denature both the sample and input DNA in a heatblock set at 100 °C for 10 minutes. Cool down on ice. When cold, add 2 μl of anti-Methylcytosine (5MeC) antibody (Diagenode, Cat#: MAb-006-100, Lot#: DA-0017) to sample DNA. Rotate tubes overnight at 4°C. Next day add 90 ul of Pro-A sepharose slurry (Pharmacia) to each tube. Rotate at 4 C for 1hr. Transfer samples and beads to a Handee spin cup column with paper filter (Pierce). Spin samples for ~10 seconds in a picofuge to remove the buffer. Wash beads for 5 min at 4 C with 400 ul each of the following buffers: low salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), high salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1), wash twice with TE (10mM Tris-HCl, 1mM EDTA, pH 8.0). After each wash, spin columns in picofuge for 10 s. After the final TE wash, elute the DNA from the beads by adding 250 l of pre-warmed (60°C) QG buffer (Qiagen Quick Spin Kit). Incubate the spin column with the QG buffer in a heat block set at 100oC for 5 min. Gently vortex the samples for 5 min at RT. Spin the sample for ~10 seconds in a picofuge and transfer the eluate to a clean 1.5 ml microfuge tube. Repeat the elution step. Add 450 μl of prewarmed QG buffer to the input DNA, incubate for 5 minutes at 100 °C, shake for 5 min. at RT, and spin in a picofuge. Add 150 μl of isopropanol to 500 μl of eluate or diluted input. Put the samples in a QIAquick column and spin for 1 minute at maximum speed. Wash with 750 ul PE buffer. Elute twice with 30 ul of PCR H2O. Vacuum concentrate.
Label
Cy3
Label protocol
DNA amplification was based on random prime synthesis using the BioPrime DNA Labeling System (Invitrogen, Carlsbad, CA). The DNA sample or equivalent amount of input in a total volume of 21 ul was mixed with 20 ul of 2.5x Random Primers Solution, denatured by heating for 5 min at 95 C in a thermal cycler, and cooled on ice for 5 min. 5 ul of 10x dUTP Nucleotide Mix, 3 ul of 1 mM dTTP and 1 ul of Exo-Klenow Fragment, were added and incubated at 37 C for 20 h. Amplified DNA was purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and quantified on a ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). The typical yield was 4-5 ug of amplified DNA per 50 ul reaction. Equal amounts (2 ug) of amplified immunoprecipitated and input DNA were labeled using the BioPrime DNA Labeling System (Invitrogen) with Cy3 or Cy5 dyes respectively. DNA in a volume of 42 ul was mixed with 40 ul 2.5x Random Primers Solution, denatured by heating for 5 min at 95 C in a thermal cycler, and cooled on ice for 5 min. 10 ul of 10x dUTP Nucleotide Mix, 4 ul of 1 mM dTTP, 2 ul of Cy3- or Cy5-dUTP (1mM) (GE healthcare, Piscataway, NJ, USA) and 2 ul of Exo-Klenow Fragment were added and the mixture was incubated for 20 h at 37 C. Labeled targets were purified using the QIAquick PCR purification kit (Qiagen). The efficiency of labeling was determined using ND-1000 spectrophotometer (Nanodrop Technologies). The typical yield was 200 - 300 pmol of dye incorporated in 7 - 8 ug of DNA per sample.
M87A medium supplemented with oxytocin and cholera toxin at 0.5 ng/ml
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). 5 μg of RNase treated DNA was dried under vacuum and resuspended with 50 μl SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and 450 μl of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl). Vortex for ~20 minutes to fully resuspend the DNA. Sonicate the samples 5x for 10 seconds (5 times total) at 8 watts. Make sure that most of the DNA is between 300-600 bp long. Set aside 50 μl of sample in a new microfuge tube - Input DNA. [immunoprecipitation protocol] Denature both the sample and input DNA in a heatblock set at 100 °C for 10 minutes. Cool down on ice. When cold, add 2 μl of anti-Methylcytosine (5MeC) antibody (Diagenode, Cat#: MAb-006-100, Lot#: DA-0017) to sample DNA. Rotate tubes overnight at 4°C. Next day add 90 ul of Pro-A sepharose slurry (Pharmacia) to each tube. Rotate at 4 C for 1hr. Transfer samples and beads to a Handee spin cup column with paper filter (Pierce). Spin samples for ~10 seconds in a picofuge to remove the buffer. Wash beads for 5 min at 4 C with 400 ul each of the following buffers: low salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), high salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1), wash twice with TE (10mM Tris-HCl, 1mM EDTA, pH 8.0). After each wash, spin columns in picofuge for 10 s. After the final TE wash, elute the DNA from the beads by adding 250 l of pre-warmed (60°C) QG buffer (Qiagen Quick Spin Kit). Incubate the spin column with the QG buffer in a heat block set at 100oC for 5 min. Gently vortex the samples for 5 min at RT. Spin the sample for ~10 seconds in a picofuge and transfer the eluate to a clean 1.5 ml microfuge tube. Repeat the elution step. Add 450 μl of prewarmed QG buffer to the input DNA, incubate for 5 minutes at 100 °C, shake for 5 min. at RT, and spin in a picofuge. Add 150 μl of isopropanol to 500 μl of eluate or diluted input. Put the samples in a QIAquick column and spin for 1 minute at maximum speed. Wash with 750 ul PE buffer. Elute twice with 30 ul of PCR H2O. Vacuum concentrate.
Label
Cy5
Label protocol
DNA amplification was based on random prime synthesis using the BioPrime DNA Labeling System (Invitrogen, Carlsbad, CA). The DNA sample or equivalent amount of input in a total volume of 21 ul was mixed with 20 ul of 2.5x Random Primers Solution, denatured by heating for 5 min at 95 C in a thermal cycler, and cooled on ice for 5 min. 5 ul of 10x dUTP Nucleotide Mix, 3 ul of 1 mM dTTP and 1 ul of Exo-Klenow Fragment, were added and incubated at 37 C for 20 h. Amplified DNA was purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and quantified on a ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). The typical yield was 4-5 ug of amplified DNA per 50 ul reaction. Equal amounts (2 ug) of amplified immunoprecipitated and input DNA were labeled using the BioPrime DNA Labeling System (Invitrogen) with Cy3 or Cy5 dyes respectively. DNA in a volume of 42 ul was mixed with 40 ul 2.5x Random Primers Solution, denatured by heating for 5 min at 95 C in a thermal cycler, and cooled on ice for 5 min. 10 ul of 10x dUTP Nucleotide Mix, 4 ul of 1 mM dTTP, 2 ul of Cy3- or Cy5-dUTP (1mM) (GE healthcare, Piscataway, NJ, USA) and 2 ul of Exo-Klenow Fragment were added and the mixture was incubated for 20 h at 37 C. Labeled targets were purified using the QIAquick PCR purification kit (Qiagen). The efficiency of labeling was determined using ND-1000 spectrophotometer (Nanodrop Technologies). The typical yield was 200 - 300 pmol of dye incorporated in 7 - 8 ug of DNA per sample.
Hybridization protocol
Combined Cy3 and Cy5 labeled samples were vaccum concentrated to volume of 91.5 µl. 12.5 µl of human Cot-1 DNA (1µg/µl, Invitrogen Cat. No. 15279-011), 26 µl of Agilent blocking agent (10x) and 130 µl of Agilent hybridization buffer (2x) were added. Samples were heated for 3 min at 95 oC, transferred to 37 oC, incubated for 30 min, and then used for microarray hybridization for 28h at 65 oC. After hybridization, slides were washed in Agilent Oligo aCGH/ChIP-on-Chip Wash Buffer 1 for 5 min at room temperature, then in Agilent Oligo aCGH/ChIP-on-Chip Wash Buffer 2 for 5 min at 37 oC, washed in acetronitrile for 10 s at room temperature and finally in stabilization and drying solution for 30 s at room temperature.
Scan protocol
Slides were scanned using an Axon GenePix 4000B microarray scanner (Axon Instruments, Inc., Foster City, CA, USA) and GenePix 6.0 software at 5 µm resolution and PMT settings 750 (635nm) and 600 (532nm)
Description
Immortal human mammary epithelial cells
Data processing
Output from GenePix (*.gpr files) were imported to R using the limma package. Individual channels were first spatially normalized within arrays using ma2D function from the package marray and then loess normalized between arrays using the function normalize.loess from package affy. The RG object was transformed to an MA object and M values were again loess normalized between arrays.
