Glass needle–based microdissection was performed on G-banded chromosomes as described earlier16 . One copy of each chromosome was collected. Chromosome-specific libraries were created with whole-genome amplification kits (Sigma). After the amplification, DNA was purified by means of nucleic acid purification kits for DNA (BioSilica). DNA libraries were labeled by whole-genome amplification kits (Sigma) according to the manufacturer's protocol. Chromosome-specific probes were created for both homologous chromosomes 13 and homologous chromosomes 14 of the female U. kamensis (Q18-96) and for chromosomes and chromosome regions of M. auratus (MAUR): 6q distal, 6q proximal, 9, 11, 13, 14, 15, and 18 distal.
Whole genome amplification kit
Manufactured by Merck Group
Sourced in United States, Canada
The Whole Genome Amplification kit is a laboratory tool used to rapidly and efficiently amplify small amounts of DNA to generate larger quantities for further analysis. The kit contains reagents and protocols that enable the simultaneous amplification of all genomic regions, providing a comprehensive representation of the entire genome.
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Lab products found in correlation
Anti 5 methylcytosine, by Eurogentec (1 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Anti acetyl histone h3 antibody, by Merck Group (1 mentions)
Nimblegen dual colordna labeling kit, by Roche (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Hydroxymethyl collector kit, by Active Motif (1 mentions)
Methylcollector kit, by Active Motif (1 mentions)
Cy3 dutp, by PerkinElmer (1 mentions)
Cy5 dutp, by PerkinElmer (1 mentions)
Microarray scanner, by Agilent Technologies (1 mentions)
Suretag complete dna labeling kit, by Agilent Technologies (1 mentions)
Pcr cleanup kit, by Qiagen (1 mentions)
Eb buffer, by Qiagen (1 mentions)
Plasmid midi kit, by Qiagen (1 mentions)
Haeiii, by New England Biolabs (1 mentions)
Medip kit, by Diagenode (1 mentions)
Maxwell 16 ffpe plus lev dna purification kit, by Promega (1 mentions)
Maxwell 16 cell lev dna purification kit, by Promega (1 mentions)
Genechip scanner gcs3000, by Thermo Fisher Scientific (1 mentions)
Fluidic station 450, by Thermo Fisher Scientific (1 mentions)
16 protocols using whole genome amplification kit
1
Chromosome-specific library generation
2
MeDIP-Seq Protocol for Methylome Analysis
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The procedure used for MeDIP analysis was adapted from previously published protocols.35 (link),38 (link) Briefly, 2 μg of DNA were sonicated, and methylated DNA was immunoprecipitated using anti-5-methyl-cytosine (Eurogentec, Fremont, CA, USA). The DNA-antibody complex was immunoprecipitated with protein G, and the methylated DNA was resuspended in digestion buffer (50 mM TRisHCl pH8; 10 mM EDTA; 0.5% SDS) and treated with proteinase K overnight at 55 °C. The input and bound fraction were purified, amplified using the Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO, USA), and labeled for microarray hybridization with Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent Technologies, Mississauga, ON, Canada) in accordance with the manufactureŕs instructions. Custom designed tiling arrays were used (Agilent Technologies). All steps of the hybridization, washing, scanning and feature extraction procedures were performed in accordance with the Agilent Technologies protocol for chip-on-chip analysis. Extracted microarray intensities were processed and analyzed using the R software environment for statistical computing (http://www.r-project.org/ ).
3
Quantitative Detection of 5-hmC
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Real-time qPCR was performed on pulldown and input DNA obtained from CLICK chemistry 5-hmC pulldown (Song et al. 2011 (link)). The DNA was amplified by the Whole Genome Amplification Kit (Sigma). A total of 100 pg of DNA was used per reaction using 5× EvaGreen Supermix (Bio-Rad) and primers for selected genes.
4
Acetyl-H3 ChIP-chip Analysis of T Cells
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The DNA immunoprecipitate samples were pooled from four type 1 diabetes patients or five healthy controls in equal quantities of CD4+ T lymphocytes in total. One pooled sample consisting of 3 million mixed CD4+ T lymphocytes was used for sonication. Sonicate cell lysate was divided into three aliquots, and one aliquot contained approximate 1 million cell equivalents of chromatin, which was used for immunoprecipitation with 5.0 μg anti‐acetyl histone H3 antibody (#06‐599; Millipore). Before the antibody was added, 10 μL of the sonicate cell lysate supernatant was removed as input. The EZ ChIP™ Chromatin Immunoprecipitation Kit (#17‐371, Millipore) was used to carry out the chromatin immunoprecipitation (ChIP) assay and DNA purification. DNA was amplified with the Whole Genome Amplification kit from Sigma‐Aldrich (Darmstadt, Germany). Fluorescent labeling of the DNA was carried out using the NimbleGen Dual‐Color DNA Labeling Kit (F. Hoffman‐La Roche Ltd., Basel, Switzerland). Each pooled sample was labeled and hybridized to Roche Nimblegen human 720K RefSeq promoter tiling arrays, with probes designed to cover –3,200–800 bp regions relative to the transcription start sites of 22,542 Refseq genes, to detect sheared DNA pulled down by acetyl‐H3 antibody at promoter regions. Hybridizations were carried out by KangChen Bio‐tech Inc. (Shanghai, China).
5
Hydroxymethyl DNA Enrichment and Profiling
6
Genome-wide DNA Methylation Profiling
7
Methylated DNA Immunoprecipitation for Microarray
8
Genome-wide Promoter Analysis by ChIP-chip
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After ChIP, the immunoprecipitated DNA was pre-amplified with a whole-genome amplification kit (Sigma #WGA2) following the manufacturer’s protocol, and 2 μg DNA was labeled using a SureTag complete DNA labeling kit (Agilent Technologies #51904240) at 37 °C for 2 h then 65 °C for 10 min. Input DNA was then labeled with cyanine 3 while immunoprecipitated DNA was labeled with cyanine 5. Both samples were purified on columns and eluted in TE buffer. Labeled DNAs were mixed and competitively hybridized to DNA microarrays. Hybridization was carried out on 2X400K Sure Print G3 Human promoter microarrays (Agilent #G4874A) in the presence of human Cot-1 DNA for 40 h at 65 °C and the slides were washed according to Agilent’s procedure. After washing, the slides were scanned using an Agilent microarray scanner, and intensity of fluorescent signals was extracted using Agilent feature extraction 11.2 software.
Each slide contained two identical arrays and each microarray contained 414,043 (60-mer) oligonucleotide probes spaced every 172 bp across promoter regions including −5.5 Kb upstream and +2.5 Kb downstream of identified transcriptional start sites (TSS). The probes covered 21,000 of the best-defined human transcripts represented as RefSeq genes.
Each slide contained two identical arrays and each microarray contained 414,043 (60-mer) oligonucleotide probes spaced every 172 bp across promoter regions including −5.5 Kb upstream and +2.5 Kb downstream of identified transcriptional start sites (TSS). The probes covered 21,000 of the best-defined human transcripts represented as RefSeq genes.
9
Euchromatic FISH Probe Preparation
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Euchromatic FISH probes were made as previously described (Buster 2013 (link); Smith 2013 (link)) from BAC clones (CHORI BACPAC Resources) as follows: X1, BACR30C13 and BACR18F10; X2, BACR20K01 and BACR35A18; 2L (1), BACR30M19 and BACR29P12; and 2L (2), BACR14I17 and BACR15P08. BAC clones were mapped and picked using the UCSC genome browser (genome.ucsd.edu ). Clones were cultured, DNA was purified using the Plasmid Midi Kit (Qiagen), and purified DNA was amplified using the Whole Genome Amplification kit (Sigma). Amplified DNA (µg) was digested using a restriction enzyme cocktail consisting of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs) overnight at 37° and then ethanol-precipitated. DNA was denatured at 100° for 1 min and 3′-end-labeled with aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche) for 2 hr at 37°. Five mM EDTA was added to terminate the reaction and, after ethanol precipitation, DNA was resuspended in 10 uL ddH2O and conjugated to fluorophores using ARES Alexa Fluor DNA labeling kits (Invitrogen) according to the manufacturer’s instructions. Probes were then cleaned using Qiagen PCR clean-up kit (Qiagen), ethanol-precipitated, and resuspended in 10 µL EB buffer (Qiagen).
10
ChIP Analysis of Centromeric Proteins
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Chromatin immuno-precipitation (ChIP) analysis was performed as previously described (Wells and Farnham 2002 (link)). Briefly, lymphoblastoid cells containing Neo3 were cross-linked in situ by adding formaldehyde to a 1% final concentration directly to the culture medium, and DNA was shared by sonication. Immunoprecipitation was performed using polyclonal antibodies against centromeric proteins CENP-A and CENP-C (Trazzi et al. 2009 (link)). Purified DNA fragments were amplified using the Whole Genome Amplification kit (Sigma-Aldrich). The labeled ChIP and total DNAs were co-hybridized to a NimbleGene Whole-Genome Tiling array (HG17Tiling Set 9, see Files S1 and S2 for details), which had an average resolution of 100 bp. DNA-binding peaks were identified by using the statistical model and methodology described at http://chipanalysis.genomecenter.ucdavis.edu/cgi-bin/tamalpais.cgi using stringent parameters for peak identification (98th percentile threshold and p < 0.0001) (Bieda et al. 2006 (link)).
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