fraction: immunoprecipitated DNA cell type: 8 cell cleavage stage embryo
Growth protocol
After two years of successful transplant, donated embryos were stored via vitrified cryopreservation method. After thawing, there were 7-9 available embryos which had uniform size and lower cell debris rate (less than 20%). And 5 of them were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method.
Extracted molecule
genomic DNA
Extraction protocol
Oocyte collection and DNA extraction Oocyte of patients and ovulation volunteers who assisted in pregnancy treatment in hospitals of Guangyi Hospital from January 2010 to December 2015 were collected. All the volunteers age ranged from 22-40 years. Under a stereomicroscope, individual eggs were placed in Tyrode's solution (Sage) using a stripping egg (about 140 μm in diameter, Cook). After the disappearance of the zona pellucida observed under a stereomicroscope, (About 0.1 mL / drop), the oocytes were placed in a buffer containing 2 μL of lysis buffer A (10 mM Tris-HCl, 1 mM EDTA, 10% SDS, 1% proteinase K, pH 8.0). After the cells were completely lysed, the cells were placed in a water bath at 37 ° C for about 1 hour to fully lyse the cells. Then the cell were centrifuged at 10,000 rpm for 2 minutes and stored at -80 ° C until use. 2 μl of the last wash droplet were took for PCR reaction blank control. Sperm collection and DNA extraction Sperm of patients who accepted IVF treatment in Guangzhou Medical Third Affiliated Hospital from January 2010 to December 2015 were collected. The patient's husband age ranged from 22-40 years, secondary to infertility without any male factor in patients with her husband's semen. Density gradient centrifugation and upstream separation were applied for collection of sperm. The differential lysis method [89] was used, and DNA was extracted by the kit procedure in combination with reagents using Genomic DNA urification Kit (Promega). 2PN and 4 cell embryos collected and DNA extraction Methods of ICSI: ICSI fertilization was performed on oocytes and spermatozoa from the above sources, and the mature egg was selected to remove the egg droplet to the center of the field of view. Hold the egg with the Holding needle, oocyte was transfer to the clearest state under microinjection needle, the polar body is located at 6,7 o'clock or 11,12 o'clock position, so that the injection of quasi-oocyte was in the middle 3 o'clock position. The sperm were pushed to the tip of the injection needle, injection needle was placed in the oocyte 3 o'clock vertically through the zona pellucida until sperm was pushed within the cytoplasm of the middle of the egg. After the eggs were injected one by one, they were repeatedly rinsed in a pre-prepared G1.5 Plus Petri dish and placed in 37 ° C, 5% CO2, 5% O2, 90% N2 incubators and cultured in G1.5 Plus droplets separately. For IVF fertilization, GIVF-plus which has been equilibrated overnight was used to prepare fertilized droplets according to the standard of 0.1 ml per microtube droplet per 3 eggs. Then this fertilized droplets were stored in incubator (37 ° C, 6% CO2) with coverage of mineral oil. Moderate amount of sperm was added to prepared fertilized dish under a microscope. And concentration was adjusted to 1.0 × 10 6 / ml. this sperm droplets were stored in incubator (37 ° C, 6% CO2) to wait for fertilization. After 3-4 hour’s pre-incubation, eggs were transferred to the fertilized dish. 2-3 eggs were added to one semen drop and this fertilized drop was returned to the embryo box for overnight cultivation. 2PN Collection: Prokaryotic cells were observed at 16-20 hours after ICSI or IVF under inverted microscope and fertilization information was recorded. Two circular structures with nucleolar precursors in the cytoplasm were the male and female pronuclei (2PN). Two polar bodies (2PB) can be seen in the perivitelline space when normal fertilization occured. 12 prokaryotic and ICSI-derived prokaryotes from 2PN-derived IVF will be collected to extract DNA. DNA extraction method was identical to oocyte genomic DNA extraction method. 2 prokaryotic embryos were cotinue to culture to D2 at 16-20 hours after ICSI or IVF. And 5 cells which had 4 cells, uniform blastomere size and lower fragments rate (less than 5%) were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method. D3 embryo collection and DNA extraction After two years of successful transplant, donated embryos were stored via vitrified cryopreservation method. After thawing, there were 7-9 available embryos which had uniform size and lower cell debris rate (less than 20%). And 5 of them were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method. morulae collection and DNA extraction The thawed D3 embryos were continue to cultured in 37 °C, 5% CO2, 5% O2, 90% N2 incubators. After overnight culturing, 6 mulberry embryos which were completely fused and with less than 20% fragmentation were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method. Collection of blastocyst ICM and TE and their DNA extraction The above thawed D3 embryos were cultured to D5 embryos, and high quality blastocysts were screened for this study. Screening of D5 high-quality blastocysts was based on Garden's grading standards: stage of inner cell mass and trophoblast were A or B blastocysts. Separation of ICM and trophoblast blastocyst was performed via mechanical methods, the capillary pipette segregate ICM and trophoblast blastocyst under stereomicroscope bluntly. The isolated ICM and TE were washed for several times in PBS and then these isolated ICM and TE were extracted with reference to the DNA extraction method of the oocyte. Genomic DNA were extracted according to the manufacturer’s instructions and sonicated to random fragments in size about 200-1000 bp. Immunoprecipitation of methylated DNA was performed using Biomag TM magnetic beads coupled mouse monoclonal antibody against 5-methylcytidine.
Label
Cy5
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com. The immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation. The total input and immunoprecipitated DNA were labeled with Cy3- and Cy5-labeled random 9-mers, respectively, and hybridized to NimbleGen Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Microarray, which is a multiplex slide with 3 identical arrays per slide, and each array includes 27,728 CpG Islands regions (from about - 2440bp to +610bp of the TSSs) totally covered by ~720,000 probes.
fraction: input DNA cell type: 8 cell cleavage stage embryo
Growth protocol
After two years of successful transplant, donated embryos were stored via vitrified cryopreservation method. After thawing, there were 7-9 available embryos which had uniform size and lower cell debris rate (less than 20%). And 5 of them were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method.
Extracted molecule
genomic DNA
Extraction protocol
Oocyte collection and DNA extraction Oocyte of patients and ovulation volunteers who assisted in pregnancy treatment in hospitals of Guangyi Hospital from January 2010 to December 2015 were collected. All the volunteers age ranged from 22-40 years. Under a stereomicroscope, individual eggs were placed in Tyrode's solution (Sage) using a stripping egg (about 140 μm in diameter, Cook). After the disappearance of the zona pellucida observed under a stereomicroscope, (About 0.1 mL / drop), the oocytes were placed in a buffer containing 2 μL of lysis buffer A (10 mM Tris-HCl, 1 mM EDTA, 10% SDS, 1% proteinase K, pH 8.0). After the cells were completely lysed, the cells were placed in a water bath at 37 ° C for about 1 hour to fully lyse the cells. Then the cell were centrifuged at 10,000 rpm for 2 minutes and stored at -80 ° C until use. 2 μl of the last wash droplet were took for PCR reaction blank control. Sperm collection and DNA extraction Sperm of patients who accepted IVF treatment in Guangzhou Medical Third Affiliated Hospital from January 2010 to December 2015 were collected. The patient's husband age ranged from 22-40 years, secondary to infertility without any male factor in patients with her husband's semen. Density gradient centrifugation and upstream separation were applied for collection of sperm. The differential lysis method [89] was used, and DNA was extracted by the kit procedure in combination with reagents using Genomic DNA urification Kit (Promega). 2PN and 4 cell embryos collected and DNA extraction Methods of ICSI: ICSI fertilization was performed on oocytes and spermatozoa from the above sources, and the mature egg was selected to remove the egg droplet to the center of the field of view. Hold the egg with the Holding needle, oocyte was transfer to the clearest state under microinjection needle, the polar body is located at 6,7 o'clock or 11,12 o'clock position, so that the injection of quasi-oocyte was in the middle 3 o'clock position. The sperm were pushed to the tip of the injection needle, injection needle was placed in the oocyte 3 o'clock vertically through the zona pellucida until sperm was pushed within the cytoplasm of the middle of the egg. After the eggs were injected one by one, they were repeatedly rinsed in a pre-prepared G1.5 Plus Petri dish and placed in 37 ° C, 5% CO2, 5% O2, 90% N2 incubators and cultured in G1.5 Plus droplets separately. For IVF fertilization, GIVF-plus which has been equilibrated overnight was used to prepare fertilized droplets according to the standard of 0.1 ml per microtube droplet per 3 eggs. Then this fertilized droplets were stored in incubator (37 ° C, 6% CO2) with coverage of mineral oil. Moderate amount of sperm was added to prepared fertilized dish under a microscope. And concentration was adjusted to 1.0 × 10 6 / ml. this sperm droplets were stored in incubator (37 ° C, 6% CO2) to wait for fertilization. After 3-4 hour’s pre-incubation, eggs were transferred to the fertilized dish. 2-3 eggs were added to one semen drop and this fertilized drop was returned to the embryo box for overnight cultivation. 2PN Collection: Prokaryotic cells were observed at 16-20 hours after ICSI or IVF under inverted microscope and fertilization information was recorded. Two circular structures with nucleolar precursors in the cytoplasm were the male and female pronuclei (2PN). Two polar bodies (2PB) can be seen in the perivitelline space when normal fertilization occured. 12 prokaryotic and ICSI-derived prokaryotes from 2PN-derived IVF will be collected to extract DNA. DNA extraction method was identical to oocyte genomic DNA extraction method. 2 prokaryotic embryos were cotinue to culture to D2 at 16-20 hours after ICSI or IVF. And 5 cells which had 4 cells, uniform blastomere size and lower fragments rate (less than 5%) were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method. D3 embryo collection and DNA extraction After two years of successful transplant, donated embryos were stored via vitrified cryopreservation method. After thawing, there were 7-9 available embryos which had uniform size and lower cell debris rate (less than 20%). And 5 of them were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method. morulae collection and DNA extraction The thawed D3 embryos were continue to cultured in 37 °C, 5% CO2, 5% O2, 90% N2 incubators. After overnight culturing, 6 mulberry embryos which were completely fused and with less than 20% fragmentation were used in present study. DNA extraction method was identical to oocyte genomic DNA extraction method. Collection of blastocyst ICM and TE and their DNA extraction The above thawed D3 embryos were cultured to D5 embryos, and high quality blastocysts were screened for this study. Screening of D5 high-quality blastocysts was based on Garden's grading standards: stage of inner cell mass and trophoblast were A or B blastocysts. Separation of ICM and trophoblast blastocyst was performed via mechanical methods, the capillary pipette segregate ICM and trophoblast blastocyst under stereomicroscope bluntly. The isolated ICM and TE were washed for several times in PBS and then these isolated ICM and TE were extracted with reference to the DNA extraction method of the oocyte. Genomic DNA were extracted according to the manufacturer’s instructions and sonicated to random fragments in size about 200-1000 bp. Immunoprecipitation of methylated DNA was performed using Biomag TM magnetic beads coupled mouse monoclonal antibody against 5-methylcytidine.
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com. The immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation. The total input and immunoprecipitated DNA were labeled with Cy3- and Cy5-labeled random 9-mers, respectively, and hybridized to NimbleGen Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Microarray, which is a multiplex slide with 3 identical arrays per slide, and each array includes 27,728 CpG Islands regions (from about - 2440bp to +610bp of the TSSs) totally covered by ~720,000 probes.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed with the Axon GenePix 4000B microarray scanner. Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
8 cell cleavage stage embryos: Patients who underwent ART, gave birth to a successful D3 embryo for more than two years, and signed an informed consent form for scientific research, 4 pieces.
Data processing
In order to avoid technical variability and evaluate methylation differences between samples, the log2-ratio getting from raw data value should be normalized. We perform Median-centering, quantile normalization and linear smoothing in this analysis process by Bioconductor packages Ringo, limma, and MEDME. After normalization, a normalized log2-ratio data (*_ratio.gff file) was created for each sample, it can be used to further peak-finding analysis. so, 'the normalized log2-ratio data' correspond to 'MeDIP/input' log2-ratio data.
