Genomic DNA was extracted from placenta tissues with premixed of controls and premixed of NTDs samples according to manufacturer’s instructions of DNeasy Blood and Tissue Kit (QIAGEN, Germany). Genomic DNA extracted from samples was quantified by Nanodrop ND-1000, and then analyzed by agarose gel electrophoresis to ensure the integrity and the quality of genomic DNA for the following experiment. After Fragmentation of the genomic DNA, the sheared DNA of each sample was quantified by Nanodrop ND-1000, and then analyzed by agarose gel electrophoresis to check the size of sheared DNA, which should between 200 – 1000 bp for the following experiment.
Label
cy5
Label protocol
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 1 placenta tissues of control fetuses
Genomic DNA was extracted from placenta tissues with premixed of controls and premixed of NTDs samples according to manufacturer’s instructions of DNeasy Blood and Tissue Kit (QIAGEN, Germany). Genomic DNA extracted from samples was quantified by Nanodrop ND-1000, and then analyzed by agarose gel electrophoresis to ensure the integrity and the quality of genomic DNA for the following experiment. After Fragmentation of the genomic DNA, the sheared DNA of each sample was quantified by Nanodrop ND-1000, and then analyzed by agarose gel electrophoresis to check the size of sheared DNA, which should between 200 – 1000 bp for the following experiment.
Label
cy3
Label protocol
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
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 from deliveries of control fetuses
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.
