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Sample GSM1220963 Query DataSets for GSM1220963
Status Public on May 29, 2014
Title Megakaryocytes vs. embryos (MvF)
Sample type genomic
 
Channel 1
Source name Megakaryocytes
Organism Mus musculus
Characteristics strain: C57BL/6
Stage: n/a
gender: male
Treatment protocol 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.
Megakaryocytes were derived and cultured as described in (Shivdasani and Schulze, 2005). Briefly, fetal livers were dissected from e13.5 C57BL/6 embryos in Hanks’ Balanced Salt Solution and placed in DMEM with 10% FBS supplemented with 100 ug/mL penicillin-streptomycin (Invitrogen). Livers were pooled based on sex of the embryo (males pooled and females pooled). To make a single cell solution, Livers were aspirated through a progression of 18G, 21G and 23G needles. To promote differentiation into megakaryocytes, cells were cultured for five days in media containing thrombopoietin (TPO; R&D Systems) at 37C with 5% CO2. Successful differentiation was identified by 1) the presence of large cells (megakaryocytes) and by 2) FACS to confirm up to 32N ploidy. Megakaryocytes were further isolated by placing cultured cells over a two-step density gradient (1.5% BSA over 3% BSA in a 15mL tube). Megakaryocytes sank to the bottom of the tube while smaller, undifferentiated, cells stayed in the upper portion.
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.
 
Channel 2
Source name Embryo - e13.5
Organism Mus musculus
Characteristics strain: C57BL/6
Stage: e13.5
gender: female
Treatment protocol 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.
Megakaryocytes were derived and cultured as described in (Shivdasani and Schulze, 2005). Briefly, fetal livers were dissected from e13.5 C57BL/6 embryos in Hanks’ Balanced Salt Solution and placed in DMEM with 10% FBS supplemented with 100 ug/mL penicillin-streptomycin (Invitrogen). Livers were pooled based on sex of the embryo (males pooled and females pooled). To make a single cell solution, Livers were aspirated through a progression of 18G, 21G and 23G needles. To promote differentiation into megakaryocytes, cells were cultured for five days in media containing thrombopoietin (TPO; R&D Systems) at 37C with 5% CO2. Successful differentiation was identified by 1) the presence of large cells (megakaryocytes) and by 2) FACS to confirm up to 32N ploidy. Megakaryocytes were further isolated by placing cultured cells over a two-step density gradient (1.5% BSA over 3% BSA in a 15mL tube). Megakaryocytes sank to the bottom of the tube while smaller, undifferentiated, cells stayed in the upper portion.
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.
 
Submission date Sep 03, 2013
Last update date May 30, 2014
Contact name Roberta L Hannibal
E-mail(s) robertah@stanford.edu
Organization name Stanford University
Department Genetics
Street address 300 Pasteur Drive
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
 
Platform ID GPL10449
Series (2)
GSE50543 Trophoblast giant cell underrepresentation (CGH)
GSE50585 Trophoblast giant cell underrepresentation

Data table header descriptions
ID_REF
VALUE Normalized Log2 ratio of test/control.

Data table
ID_REF VALUE
107727 -0.14691457
12603 -0.195367066
150389 0.181328641
28542 0.188467296
45744 0.11394953
43647 0.058530551
106336 0.066090957
33635 0.22121712
69025 -0.002394925
156371 -0.145779842
172930 0.194852481
121051 0.088527246
65236 -0.020594946
71850 -0.083147959
48015 -0.033231215
77199 -0.091092928
160308 0.134100103
61971 -0.06088659
13072 0.219695066
54347 0.066827908

Total number of rows: 174305

Table truncated, full table size 3188 Kbytes.




Supplementary file Size Download File type/resource
GSM1220963_US85103615_252741110295_S01_CGH_1010_Sep10_1_2.txt.gz 18.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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