Pregnant C57BL/6 mice were obtained from Charles River. Copulation was determined by the presence of a vaginal plug the morning after mating, and embryonic day 0.5 (e0.5) was defined as noon of that day. TGCs and embryos were dissected in 1X PBS (1:10 10X PBS, pH=7.4; Gibco) and stored on ice until further processing. TGCs were dissected away from the placental disk, decidua, and, if possible, Reichert’s membrane. For gathering 2N genomic DNA, at e9.5-10.5, the embryo body, after removal of obvious organs and head (removed at otic vesicle), was collected; and at later stages, limbs, or a mixture of limbs and the tail, were collected. All experimental procedures were carried out in accordance with the APLAC protocol and the institutional guidelines set by the Veterinary Service Center at Stanford University.
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.
Pregnant C57BL/6 mice were obtained from Charles River. Copulation was determined by the presence of a vaginal plug the morning after mating, and embryonic day 0.5 (e0.5) was defined as noon of that day. TGCs and embryos were dissected in 1X PBS (1:10 10X PBS, pH=7.4; Gibco) and stored on ice until further processing. TGCs were dissected away from the placental disk, decidua, and, if possible, Reichert’s membrane. For gathering 2N genomic DNA, at e9.5-10.5, the embryo body, after removal of obvious organs and head (removed at otic vesicle), was collected; and at later stages, limbs, or a mixture of limbs and the tail, were collected. All experimental procedures were carried out in accordance with the APLAC protocol and the institutional guidelines set by the Veterinary Service Center at Stanford University.
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.
