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| Status |
Public on Jun 10, 2014 |
| Title |
PAR2/FOR2_2 (MeDip) |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
MeDIP ChIP DNA from Camarhynchus parvulus (PAR) erythrocytes
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| Organism |
Camarhynchus parvulus |
| Characteristics |
species (symbol): Camarhynchus parvulus (PAR)
tissue: blood
cell type: erythrocytes
antibody: anti-5-methylCytidine
antibody manufacturer: Diagenode
|
| Treatment protocol |
Experimental
|
| Growth protocol |
Birds were captured from January-April 2009 at El Garrapatero, a lowland arid site on Santa Cruz Island, Galapagos Archipelago, Ecuador, with mist nests and banded with numbered Monel bands to track recaptures. Birds were identified, aged and sexed using size and plumage characteristics.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
A small blood sample (90 µl) from each bird was collected in a microcapillary tube via brachial venipuncture. Samples were stored on wet ice in the field, then erythrocytes purified by centrifugation and cells stored in a -20°C freezer at a field station. Following the field season, samples were placed in a -80°C freezer for longer term storage.Erythrocyte DNA was isolated with DNAeasy Blood and Tissue Kit (Qiagen, Valencia, CA) and then stored at -80°C prior to analysis. DNA was sonicated as following: DNA suspension was centrifuged at 500 g for 5 min. The DNA was then resuspended in 5 ml NIM free of EDTA, PMSF, and leupeptin and centrifuged again. A small amount of DNA suspension (1 µl) was finally suspended in 5 µl of EDTA- and protease inhibitor-free NIM medium containing 12% polyvinyl pyrrolidone (PVP; Mr 360 000) (without protease inhibitors) (Tateno et al. 2000) and then purified using a series of washes and centrifugations (Ward et al. 1999) from variable number of animals per species analyzed. The same concentrations of DNA from individual blood samples were then used to produce pools of DNA material. Two DNA pools were produced in total per species, each one containing the same amount of DNA from different animals. The number of individuals used per pool is shown in the manuscript's Supplemental Table S6. These DNA pools were then used for chromatin immunoprecipitation of methylated DNA fragments (MeDIP). MeDIP was performed as follows: 6 µg of genomic DNA was subjected to series of three 20 pulse sonications at 20% amplitude and the appropriate fragment size (200-1000 ng) was verified through 2% agarose gels; the sonicated genomic DNA was resuspended in 350 µl TE buffer and denatured for 10 min at 95°C and then immediately placed on ice for 5 min; 100 µl of 5X IP buffer (50 mM Na-phosphate pH 7, 700 mM NaCl, 0.25% Triton X-100) was added to the sonicated and denatured DNA. An overnight incubation of the DNA was performed with 5 µg of antibody anti-5-methylCytidine monoclonal from Diagenode (Denville, NJ) at 4°C on a rotating platform. Protein A/G beads from Santa Cruz were prewashed on PBS-BSA 0.1% and resuspended in 40 µl 1X IP buffer. Beads were then added to the DNA-antibody complex and incubated 2 h at 4°C on a rotating platform. Beads bound to DNA-antibody complex were washed 3 times with 1 ml 1X IP buffer; washes included incubation for 5 min at 4°C on a rotating platform and then centrifugation at 6000 rpm for 2 min. Beads DNA-antibody complex were then resuspended in 250 µl digestion buffer (50 mM Tris HCl pH 8, 10 mM EDTA, 0.5% SDS) and 3.5 µl of proteinase K (20 mg/ml) was added to each sample and then incubated overnight at 55°C on a rotating platform. DNA purification was performed first with phenol and then with chloroform:isoamyl alcohol. Two washes were then performed with 70% ethanol, 1 M NaCl and glycogen. MeDIP selected DNA was then resuspended in 30 µl TE buffer.
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| Label |
Cy5
|
| Label protocol |
For one sub-array of each species, MeDIP DNA samples from the experimental groups were labeled with Cy5 and MeDIP DNA samples from the control lineage were labeled with Cy3. For the other sub-array of each species a dye swap was performed so that MeDIP DNA samples from the experimental groups were labeled with Cy3 and MeDIP DNA samples from the control lineage were labeled with Cy5.
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| Channel 2 |
| Source name |
MeDIP ChIP DNA from Geospiza fortis (FOR) erythrocytes
|
| Organism |
Geospiza fortis |
| Characteristics |
species (symbol): Geospiza fortis (FOR)
tissue: blood
cell type: erythrocytes
antibody: anti-5methylCytidine
antibody manufacturer: Diagenode
|
| Treatment protocol |
Control
|
| Growth protocol |
Birds were captured from January-April 2009 at El Garrapatero, a lowland arid site on Santa Cruz Island, Galapagos Archipelago, Ecuador, with mist nests and banded with numbered Monel bands to track recaptures. Birds were identified, aged and sexed using size and plumage characteristics.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A small blood sample (90 µl) from each bird was collected in a microcapillary tube via brachial venipuncture. Samples were stored on wet ice in the field, then erythrocytes purified by centrifugation and cells stored in a -20°C freezer at a field station. Following the field season, samples were placed in a -80°C freezer for longer term storage.Erythrocyte DNA was isolated with DNAeasy Blood and Tissue Kit (Qiagen, Valencia, CA) and then stored at -80°C prior to analysis. DNA was sonicated as following: DNA suspension was centrifuged at 500 g for 5 min. The DNA was then resuspended in 5 ml NIM free of EDTA, PMSF, and leupeptin and centrifuged again. A small amount of DNA suspension (1 µl) was finally suspended in 5 µl of EDTA- and protease inhibitor-free NIM medium containing 12% polyvinyl pyrrolidone (PVP; Mr 360 000) (without protease inhibitors) (Tateno et al. 2000) and then purified using a series of washes and centrifugations (Ward et al. 1999) from variable number of animals per species analyzed. The same concentrations of DNA from individual blood samples were then used to produce pools of DNA material. Two DNA pools were produced in total per species, each one containing the same amount of DNA from different animals. The number of individuals used per pool is shown in the manuscript's Supplemental Table S6. These DNA pools were then used for chromatin immunoprecipitation of methylated DNA fragments (MeDIP). MeDIP was performed as follows: 6 µg of genomic DNA was subjected to series of three 20 pulse sonications at 20% amplitude and the appropriate fragment size (200-1000 ng) was verified through 2% agarose gels; the sonicated genomic DNA was resuspended in 350 µl TE buffer and denatured for 10 min at 95°C and then immediately placed on ice for 5 min; 100 µl of 5X IP buffer (50 mM Na-phosphate pH 7, 700 mM NaCl, 0.25% Triton X-100) was added to the sonicated and denatured DNA. An overnight incubation of the DNA was performed with 5 µg of antibody anti-5-methylCytidine monoclonal from Diagenode (Denville, NJ) at 4°C on a rotating platform. Protein A/G beads from Santa Cruz were prewashed on PBS-BSA 0.1% and resuspended in 40 µl 1X IP buffer. Beads were then added to the DNA-antibody complex and incubated 2 h at 4°C on a rotating platform. Beads bound to DNA-antibody complex were washed 3 times with 1 ml 1X IP buffer; washes included incubation for 5 min at 4°C on a rotating platform and then centrifugation at 6000 rpm for 2 min. Beads DNA-antibody complex were then resuspended in 250 µl digestion buffer (50 mM Tris HCl pH 8, 10 mM EDTA, 0.5% SDS) and 3.5 µl of proteinase K (20 mg/ml) was added to each sample and then incubated overnight at 55°C on a rotating platform. DNA purification was performed first with phenol and then with chloroform:isoamyl alcohol. Two washes were then performed with 70% ethanol, 1 M NaCl and glycogen. MeDIP selected DNA was then resuspended in 30 µl TE buffer.
|
| Label |
Cy3
|
| Label protocol |
For one sub-array of each species, MeDIP DNA samples from the experimental groups were labeled with Cy5 and MeDIP DNA samples from the control lineage were labeled with Cy3. For the other sub-array of each species a dye swap was performed so that MeDIP DNA samples from the experimental groups were labeled with Cy3 and MeDIP DNA samples from the control lineage were labeled with Cy5.
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| Hybridization protocol |
A DNA methylated custom array by Roche Nimblegen that consisted of a Whole Genome Tiling Array of Zebra finch (Taeniopygia guttata) made of four 2.1M and one 3x720k array with 8,539,570 probes per array. Probe sizes were 50-75mer in length and median probe spacing was 200bp. Two different comparative (MeDIP vs MeDIP) hybridizations experiment were performed (2 sub-arrays) for each experimental species (FUL, SCA, PAR, CRA) versus control FOR, with each sub-array including hybridizations from MeDIP DNA from DNA pools from these different species.
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| Scan protocol |
Scanning and image acquisition was performed in-house by Nimblegen Inc.
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| Description |
Camarhynchus parvulus-PAR (experimental) sample 2 is comapred to Geospiza fortis-FOR (control) sample 2
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| Data processing |
For each comparative hybridization experiment raw data from both the Cy3 and Cy5 channels were imported into R, checked for quality and converted into MA values. Within each array, probes were separated into groups by GC content and each group was separately normalized using the loess normalization procedure. This allowed for groups with optimal GC content, which exhibited a reduced quality issue, to receive a normalization curve specific to that group. After each array was normalized within array, the arrays were then normalized across arrays using the A-quantile normalization procedure (Guerrero-Bosagna et al. 2010). Following normalization each adjacent ≥3 probe set value represents the median intensity difference between FUL, SCA, PAR and CRA and control FOR of a 600bp window. Significance was assigned to probe differences between experimental species samples and reference FOR samples by calculating the median value of the intensity differences as compared to a normal distribution scaled to the experimental mean and standard deviation of the normalized data. A Z-score and P-value were computed for each probe from that distribution. The statistically significant differential DNA methylation regions (DMR) were identified and p-values associated with each region represented. Each region of interest was then annotated for gene and CpG content. Regions chosen for validation were then described as overlapping regions of interest between the 2 hybridization experiments. This list was further reduced to those overlapping regions with an average intensity value exceeding 9.5 (log scale), at least one 100 bp region with two CpGs or a CpG density>1. (Guerrero-Bosagna et al. 2010). The July 2008 assembly of the Zebra Finch genome (taeGut1, WUSTL v3.2.4) produced by the Genome Sequencing Center at the Washington University in St. Louis (WUSTL) School of Medicine was retrieved (WUSTL 2008). A seed file was constructed and a BSgenome package was forged for using the Finch DNA sequence in the R code (Herve Pages BSgenome: Infrastructure for Biostrings-based genome data packages. R package version 1.24.0). This sequence was used to design the custom tiling arrays and to perform the bioinformatics.The chromosomal location of CNV and DMR clusters used an R-code developed to find chromosomal locations of clusters (M. K. Skinner et al. 2012). A 2 Mb sliding window with 50,000 base intervals was used to find the associated CNV and DMR in each window. A Z-test statistical analysis with p<0.05 was used on these windows to find the ones with over-represented CNV and DMR were merged together to form clusters. A typical cluster region averaged approximately 3 megabases in size. The DMR and CNV association with specific zebra finch genes and genome locations used the Gene NCBI database for zebra finch gene locations and correlated the epimutations associated (overlapped) with the genes. The three adjacent probes constituted approximately a 200bp homology search. The KEGG pathway associations were identified by using the KEGG website 'Search Pathway' tool (M. K. Skinner et al. 2012). Statistically significant over-representation uses a Fisher’s exact analysis. All DMR and CNV genomic data obtained in the current study have been deposited in the NCBI public GEO database. Spearman Rank correlation coefficients were used to test for a relationship between phylogenetic distance and epigenetic and genetic changes. Spearman Rank correlation coefficients were used to test for a relationship between phylogenetic distance and epigenetic and genetic changes (Whitlock and Schluter 2009).
R-code data processing of raw data as described was used as the basis for conclusions in this study. Value definitions for processed data files: #ClusterID = ID and chromosomal location for cluster of probes showing a significant difference (p<1.0E-5) between red and green channel hybridization (treated vs. control), #Chromosome = Chromosome number, #cSTART = Cluster start site, #cSTOP = Cluster stop site, #meanM = mean log2 intensity ratio (treated vs. control) across all microarray chips for this treatment/control group, #meanA = mean log2 hybridization intensity (both red and green channel) across all microarray chips for this treatment/control group, #minP = p-value for difference between treated and control intensity as calculated by indicated R-code from raw data files. P-value must be p<1.0E-5 or less for inclusion, #nProbes = number of consecutive probes with p<1.0E-5 included in cluster, #GC = GC content of cluster, #CpG = CpG content of cluster, #CpGdensity = number of CpGs per hundred base pairs, and #Seq = probe sequence of cluster.
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| Submission date |
Jun 09, 2014 |
| Last update date |
Jun 12, 2014 |
| Contact name |
Michael K Skinner |
| E-mail(s) |
skinner@mail.wsu.edu
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| Organization name |
WSU
|
| Department |
SBS
|
| Street address |
Abelson 507
|
| City |
Pullman |
| State/province |
WA |
| ZIP/Postal code |
99163 |
| Country |
USA |
| |
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| Platform ID |
GPL18772 |
| Series (1) |
| GSE58334 |
Epigenetics and the Evolution of Darwin’s Finches |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1407374_546257A01_532.pair.gz |
76.0 Mb |
(ftp)(http) |
PAIR |
| GSM1407374_546257A01_635.pair.gz |
76.6 Mb |
(ftp)(http) |
PAIR |
| GSM1407374_546257A01_ratio.gff.gz |
59.0 Mb |
(ftp)(http) |
GFF |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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