Genomic DNA (gDNA) was extracted from 6 Cell samples using a DNeasy Blood & Tissue Kit (Qiagen, Fremont, CA). The purified gDNA was then quantified and quality assessed by nanodrop ND-1000. Genomic DNA of each sample was sonicated to ~200 – 1000 bp with a Bioruptor sonicator (Diagenode) on “Low” mode for 10 cycles of 30 seconds “ON” & 30 seconds “OFF”. The gDNA and each sheared DNA were aragrose analyzed. 1 μg of sonicated genomic DNA was used for immunoprecipitation using a mouse monoclonal anti-5-methylcytosine antibody (Diagenode). For this, DNA was heat-denatured at 94 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4 °C with rocking agitation in 400 μL immunoprecipitation buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 200 μL of anti-mouse IgG magnetic beads were added and incubated for an additional 2 hours at 4 °C with agitation. After immunoprecipitation, a total of five immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25mg/mL proteinase K for 2 hours at 65°C and then allowed to cool down to room temperature. MeDIP DNA were purified using Qiagen MinElute columns (Qiagen)Immunoprecipitation of methylated DNA was performed using BiomagTM magnetic beads coupled mouse monoclonal antibody against 5-methylcytidine. The immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation. The MeDIP-enriched DNA was amplified using a WGA kit from Sigma-Aldrich (GenomePlex® Complete Whole Genome Amplification (WGA2) kit). The amplified DNA samples were then purified with QIAquick PCR purification kit (Qiagen).
Label
Cy5
Label protocol
The purified DNA was quantified using a nanodrop ND-1000. For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen MeDIP-chip protocol (Nimblegen Systems, Inc., Madison, WI, USA). 1 μg DNA of each sample was incubated for 10 min at 98°C with 1 OD of Cy5-9mer primer (IP sample) or Cy3-9mer primer (Input sample). Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled DNA was purified by isopropanol / ethanol precipitation.
Channel 2
Source name
Input DNA from subject 2 sperm sample after selection
Genomic DNA (gDNA) was extracted from 6 Cell samples using a DNeasy Blood & Tissue Kit (Qiagen, Fremont, CA). The purified gDNA was then quantified and quality assessed by nanodrop ND-1000. Genomic DNA of each sample was sonicated to ~200 – 1000 bp with a Bioruptor sonicator (Diagenode) on “Low” mode for 10 cycles of 30 seconds “ON” & 30 seconds “OFF”. The gDNA and each sheared DNA were aragrose analyzed. 1 μg of sonicated genomic DNA was used for immunoprecipitation using a mouse monoclonal anti-5-methylcytosine antibody (Diagenode). For this, DNA was heat-denatured at 94 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4 °C with rocking agitation in 400 μL immunoprecipitation buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 200 μL of anti-mouse IgG magnetic beads were added and incubated for an additional 2 hours at 4 °C with agitation. After immunoprecipitation, a total of five immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25mg/mL proteinase K for 2 hours at 65°C and then allowed to cool down to room temperature. MeDIP DNA were purified using Qiagen MinElute columns (Qiagen)Immunoprecipitation of methylated DNA was performed using BiomagTM magnetic beads coupled mouse monoclonal antibody against 5-methylcytidine. The immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation. The MeDIP-enriched DNA was amplified using a WGA kit from Sigma-Aldrich (GenomePlex® Complete Whole Genome Amplification (WGA2) kit). The amplified DNA samples were then purified with QIAquick PCR purification kit (Qiagen).
Label
Cy3
Label protocol
The purified DNA was quantified using a nanodrop ND-1000. For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen MeDIP-chip protocol (Nimblegen Systems, Inc., Madison, WI, USA). 1 μg DNA of each sample was incubated for 10 min at 98°C with 1 OD of Cy5-9mer primer (IP sample) or Cy3-9mer primer (Input sample). Then, 100 pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled DNA was purified by isopropanol / ethanol precipitation.
Hybridization protocol
hybridized to NimbleGen Human DNA Methylation 3x720K Promoter Plus CpG Island Arrays, which is a multiplex slide with 3 identical arrays per Microarrays were hybridized at 42°C during 16 to 20h with Cy3/5 labelled DNA in Nimblegen hybridization buffer/ hybridization component A in a hybridization chamber (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the Nimblegen Wash Buffer kit (Nimblegen Systems, Inc., Madison, WI, USA). For array hybridization, Roche NimbleGen's Human Promoter plus CpG Island array was used, which is a 3x720k format array design containing 27,728 CpG Islands and all well-characterized Promoter regions (from about -2,440bp to +610bp of the TSSs) totally covered by ~720,000 probes.
Scan protocol
Scanning was performed with the Axon GenePix 4000B microarray scanner.
Description
MeDIP-ChIP human sperm DNA after density selection
Data processing
Raw data was extracted as pair files by NimbleScan software. We perform Median-centering, quantile normalization, and linear smoothing by Bioconductor packages Ringo, limma, and MEDME. After normalization, a normalized log2-ratio data (*_ratio.gff file) was created for each sample.
