|
| Status |
Public on May 04, 2017 |
| Title |
lean epigenome, 5-hydroxymethylcytosine (5hmC) by hMeDIP |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
5hmC hMeDIP placental gDNA from lean pregnancies
|
| Organism |
Homo sapiens |
| Characteristics |
condition: lean (healthy weight) pregnancies with a mean BMI of 23.4
pregnancy complications: none
blood pressure: normotensive
neonatal outcome: normal
tissue: full-term placenta
antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from each tissue sample using DNeasy Tissue and Blood Kit (Qiagen) according to the manufacturer’s instructions, followed by ethanol reprecipitation. The quality and concentration of isolated genomic DNA was evaluated using NanoDrop 2000 (Thermo Fisher Scientific) and each DNA sample was routinely assessed by agarose gel electrophoresis with ethidium bromide staining to ascertain the absence of contaminating RNA and to determine the extent of degradation of genomic DNA preparations. Villous placental genomic DNA samples derived from 10 obese (prepregnancy or first trimester BMI with a mean and SD values of 34.0 ± 2.9) and 10 healthy weight (BMI of 23.4 ± 2.3) women with uncomplicated pregnancy were combined, resulting in two pools of DNA (each 5 male and 5 female placentas) that were subjected to methylated/hydroxymethylated DNA immunoprecipitation (MeDIP/hMeDIP) assays.
|
| Label |
Cy3
|
| Label protocol |
Fragmented genomic DNA (10 ug) was denatured and immunoprecipitated using the mouse monoclonal antibody against 5-methylcytosine (clone 33D3, Eurogentec) or rabbit polyclonal antibody against 5-hydroxymethylcytosine (Diagenode). A portion of the fragmented DNA (20%) was left untreated to serve as input control. Immunoprecipitated and input DNA were amplified using GenomePlex Complete Whole Genome Amplification (WGA2) Kit (Sigma-Aldrich). After cleanup with QIAquick PCR Purification columns (Qiagen), immunoprecipitated and input DNA were denatured and differentially labeled with fluorescent Cy5 and Cy3 dyes, respectively, using Dual-Color DNA Labeling Kit (Roche NimbleGen).
|
| |
|
| Channel 2 |
| Source name |
5hmC hMeDIP placental gDNA from lean pregnancies
|
| Organism |
Homo sapiens |
| Characteristics |
condition: lean (healthy weight) pregnancies with a mean BMI of 23.4
pregnancy complications: none
blood pressure: normotensive
neonatal outcome: normal
tissue: full-term placenta
antibody: 5-hydroxymethylcytosine (5-hmC) pAb (Diagenode)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from each tissue sample using DNeasy Tissue and Blood Kit (Qiagen) according to the manufacturer’s instructions, followed by ethanol reprecipitation. The quality and concentration of isolated genomic DNA was evaluated using NanoDrop 2000 (Thermo Fisher Scientific) and each DNA sample was routinely assessed by agarose gel electrophoresis with ethidium bromide staining to ascertain the absence of contaminating RNA and to determine the extent of degradation of genomic DNA preparations. Villous placental genomic DNA samples derived from 10 obese (prepregnancy or first trimester BMI with a mean and SD values of 34.0 ± 2.9) and 10 healthy weight (BMI of 23.4 ± 2.3) women with uncomplicated pregnancy were combined, resulting in two pools of DNA (each 5 male and 5 female placentas) that were subjected to methylated/hydroxymethylated DNA immunoprecipitation (MeDIP/hMeDIP) assays.
|
| Label |
Cy5
|
| Label protocol |
Fragmented genomic DNA (10 ug) was denatured and immunoprecipitated using the mouse monoclonal antibody against 5-methylcytosine (clone 33D3, Eurogentec) or rabbit polyclonal antibody against 5-hydroxymethylcytosine (Diagenode). A portion of the fragmented DNA (20%) was left untreated to serve as input control. Immunoprecipitated and input DNA were amplified using GenomePlex Complete Whole Genome Amplification (WGA2) Kit (Sigma-Aldrich). After cleanup with QIAquick PCR Purification columns (Qiagen), immunoprecipitated and input DNA were denatured and differentially labeled with fluorescent Cy5 and Cy3 dyes, respectively, using Dual-Color DNA Labeling Kit (Roche NimbleGen).
|
| |
|
| |
| Hybridization protocol |
Equal amounts (34 ug) of Cy5- and Cy3-labeled samples were combined and hybridized to each Human DNA Methylation 2.1M v2 genome tilling array (Roche NimbleGen) for 16-20 h at 42˚C using NimbleGen hybridization System 4. After washing, the hybridized arrays were dried using ArrayIt high-speed microarray centrifuge.
|
| Scan protocol |
Arrays were scanned on NimbleGen MS 200 microarray scanner using NimbleGen Data Collection software.
|
| Description |
uncomplicated pregnancies
hMeDIP 5hmC human normal epigenome
|
| Data processing |
Arrays were processed using the standard protocol (NimbleGen Arrays User’s Guide for DNA methylation analysis v1.0; scaled, log2 (MeDIP/input) or (hMeDIP/input) ratio in gff files).
|
| |
|
| Submission date |
Jun 11, 2015 |
| Last update date |
May 04, 2017 |
| Contact name |
Kohzoh Mitsuya |
| E-mail(s) |
mitsuya@uthscsa.edu
|
| Phone |
210-567-7064
|
| Organization name |
University of Texas Health Science Center San Antonio
|
| Department |
Center for Pregnancy and Newborn Research
|
| Lab |
Leslie Myatt
|
| Street address |
7703 Floyd Curl Drive
|
| City |
San Antonio |
| State/province |
Texas |
| ZIP/Postal code |
78229-3900 |
| Country |
USA |
| |
|
| Platform ID |
GPL16284 |
| Series (1) |
| GSE69769 |
MeDIP and hMeDIP assays using human placentas of obese and lean (healthy weight) pregnancies |
|
