Mouse TSCs were obtained from Dr. Janet Rossant, Hospital for Sick Children, (Toronto, Canada) and maintained in DMEM/F12 with 15 mM Hepes, 20% FBS, 2 mM glutamine, 100 ug/ml streptomycin, 1 mM sodium pyruvate, 100 uM BME, supplemented with Activin, FGF4, and Heparin following (Erlebacher et al, Developmental Biology 2004). Trophoblast stem cells were differentiated into trophoblast giant cells by replacing the FGF, Activin and Heparin in the media with retinoic acid (Yan et al., 2001; Erlebacher et al., 2004). Mature TGCs are seen after 4-6 days of differentiation (Carney et al., 1993). Cells were trypsinized for 4 minutes before the extract protocol. The embryonic stem cell line CGR8 is a germ-line competent cell line established from the inner cell mass of a 129 e3.5 male pre-implantation embryo (Nichols et al., 1990). Cells were cultured feeder-free on 0.1% gelatin coated plates. The ES cell medium was prepared by supplementing knockout DMEM (Invitrogen) with 15% FBS, 1mM glutamax, 0.1mM nonessential amino acids, 1mM sodium pyruvate, 0.1mM 2-mercaptoethanol, penicillin/streptomycin, and 1000 units of leukemia inhibitory factor (LIF; Millipore). Cell culture was maintained at 37°C with 5% CO2.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from fresh tissue and cultured cells using the DNeasy Blood & Tissue Kit (Qiagen). Before column purification, we digested in vivo and in vitro samples with proteinase-K (600 mAU/ml solution or 40 mAU/mg protein) overnight and for 10 minutes, respectively, at 56C, followed by 4 minute incubation with RNase A (100 mg/mL) (Qiagen DNeasy Blood & Tissue Kit). For arrays performed on DNA from TGCs and embryonic controls, genomic DNA from two individuals in the same litter were pooled for each condition. For megakaryocyte arrays, cells derived from 5-6 livers from a single litter were pooled for each condition. For controls for the megakaryocyte array, three embryos (subset of the litter from which livers were collected from) were pooled for each condition. For arrays performed on DNA from cultured cells, two replicates from different passages were used (5 million cells each).
Label
Cy5
Label protocol
For each condition, approximately 4 μg DNA was sent to the Biomedical Genomics Core at the Research Institute at Nationwide Children’s Hospital (Columbus, OH) for processing with the SurePrint G3 Mouse CGH Microarray Kit, 4x180k (Agilent). When possible (ie: all arrays performed on DNA from in vivo tissue), to ensure that the arrays detect copy number variation, duplicates consist of 1) female test versus male control and 2) male test versus female control.
Mouse TSCs were obtained from Dr. Janet Rossant, Hospital for Sick Children, (Toronto, Canada) and maintained in DMEM/F12 with 15 mM Hepes, 20% FBS, 2 mM glutamine, 100 ug/ml streptomycin, 1 mM sodium pyruvate, 100 uM BME, supplemented with Activin, FGF4, and Heparin following (Erlebacher et al, Developmental Biology 2004). Trophoblast stem cells were differentiated into trophoblast giant cells by replacing the FGF, Activin and Heparin in the media with retinoic acid (Yan et al., 2001; Erlebacher et al., 2004). Mature TGCs are seen after 4-6 days of differentiation (Carney et al., 1993). Cells were trypsinized for 4 minutes before the extract protocol. The embryonic stem cell line CGR8 is a germ-line competent cell line established from the inner cell mass of a 129 e3.5 male pre-implantation embryo (Nichols et al., 1990). Cells were cultured feeder-free on 0.1% gelatin coated plates. The ES cell medium was prepared by supplementing knockout DMEM (Invitrogen) with 15% FBS, 1mM glutamax, 0.1mM nonessential amino acids, 1mM sodium pyruvate, 0.1mM 2-mercaptoethanol, penicillin/streptomycin, and 1000 units of leukemia inhibitory factor (LIF; Millipore). Cell culture was maintained at 37°C with 5% CO2.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from fresh tissue and cultured cells using the DNeasy Blood & Tissue Kit (Qiagen). Before column purification, we digested in vivo and in vitro samples with proteinase-K (600 mAU/ml solution or 40 mAU/mg protein) overnight and for 10 minutes, respectively, at 56C, followed by 4 minute incubation with RNase A (100 mg/mL) (Qiagen DNeasy Blood & Tissue Kit). For arrays performed on DNA from TGCs and embryonic controls, genomic DNA from two individuals in the same litter were pooled for each condition. For megakaryocyte arrays, cells derived from 5-6 livers from a single litter were pooled for each condition. For controls for the megakaryocyte array, three embryos (subset of the litter from which livers were collected from) were pooled for each condition. For arrays performed on DNA from cultured cells, two replicates from different passages were used (5 million cells each).
Label
Cy3
Label protocol
For each condition, approximately 4 μg DNA was sent to the Biomedical Genomics Core at the Research Institute at Nationwide Children’s Hospital (Columbus, OH) for processing with the SurePrint G3 Mouse CGH Microarray Kit, 4x180k (Agilent). When possible (ie: all arrays performed on DNA from in vivo tissue), to ensure that the arrays detect copy number variation, duplicates consist of 1) female test versus male control and 2) male test versus female control.
Hybridization protocol
The standard steps of the Agilent arrayCGH hybridization protocol were performed by the Biomedical Genomics Core at the Research Institute at Nationwide Children’s Hospital (Columbus, OH) using the SurePrint G3 Mouse CGH Microarray Kit, 4x180k (Agilent).
Scan protocol
The standard steps of the Agilent arrayCGH scan protocol were performed by the Biomedical Genomics Core at the Research Institute at Nationwide Children’s Hospital (Columbus, OH) using the SurePrint G3 Mouse CGH Microarray Kit, 4x180k (Agilent).
Data processing
The standard steps of the Agilent arrayCGH data processing were performed by the Biomedical Genomics Core at the Research Institute at Nationwide Childrens Hospital (Columbus, OH). Log ratios from the data were then transformed into normalized log 2 ratios.
