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Cold Spring Harbor Protocols

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https://www.readbyqxmd.com/read/27852843/dna-bisulfite-sequencing-for-single-nucleotide-resolution-dna-methylation-detection
#1
Paul M Lizardi, Qin Yan, Narendra Wajapeyee
DNA methylation plays an important role in multiple biological processes. Therefore, methodologies that can detect changes in DNA methylation are of general importance. A popular and reliable method for measuring DNA methylation status is DNA bisulfite sequencing. This protocol details the steps required for bisulfite conversion and analysis of either genes or a specific genomic region. Denatured DNA (i.e., single-stranded DNA) is treated with sodium bisulfite under conditions that preferentially convert unmethylated cytosine (C) residues to uracil (U) residues while methylated cytosines remain unmodified...
November 16, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27852842/high-throughput-deep-sequencing-for-mapping-mammalian-dna-methylation
#2
Paul M Lizardi, Qin Yan, Narendra Wajapeyee
This protocol describes methylation mapping analysis by paired-end sequencing (Methyl-MAPS). In addition to the sequence information, paired-end sequencing provides information about the physical distance between the two reads in the genome. Methyl-MAPS typically samples ∼80% of the CpG dinucleotides in the genome and is also able to report the methylation status of individual genomic loci harboring repetitive elements. This is achieved by enzymatic fractionation of the genome into methylated and unmethylated compartments...
November 16, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27852841/illumina-sequencing-of-bisulfite-converted-dna-libraries
#3
Paul M Lizardi, Qin Yan, Narendra Wajapeyee
Here we describe a standard MethylC-seq protocol using single-read sequencing on an Illumina Genome Analyzer II platform. The protocol involves ligation of methylated sequencing adaptors to sonicated genomic DNA, gel purification, sodium bisulfite conversion, polymerase chain reaction (PCR) amplification, and sequencing.
November 16, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27852840/methyl-cytosine-based-immunoprecipitation-for-dna-methylation-analysis
#4
Paul M Lizardi, Qin Yan, Narendra Wajapeyee
In mammalian cells, DNA methylation at the 5-position of cytosine leads to recruitment of proteins that selectively recognize and bind 5-methylcytosine (5mC). Taking advantage of the structural identity of 5mC, various affinity purification-based protocols have been developed to enrich for either DNA that is modified by 5mC or proteins that recognize 5mC. In this protocol, an antibody against 5mC is used to immunoprecipitate the methylated DNA. The method can be scaled up to perform genome-wide DNA methylation analysis...
November 16, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27852839/analysis-of-dna-methylation-in-mammalian-cells
#5
Paul M Lizardi, Qin Yan, Narendra Wajapeyee
Methylation of DNA, the most experimentally accessible epigenetic alteration of eukaryotic cells, has generated an extensive literature and an abundance of analytical tools. The term "methylome" (referring to the complete set of cytosine modifications in a genome) is appearing with greater frequency in the literature, reflecting the growing number of researchers in the field. Here we introduce a set of robust protocols for methods that can be performed routinely for the elucidation of DNA chemical modifications involving methylation of cytosine...
November 16, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27852838/methylation-specific-polymerase-chain-reaction-pcr-for-gene-specific-dna-methylation-detection
#6
Paul M Lizardi, Qin Yan, Narendra Wajapeyee
Methylation-specific polymerase chain reaction (MS-PCR) is a more rapid way to detect changes in DNA methylation than is bisulfite sequencing. In addition, by incorporating some basic automation, samples can be prepared and analyzed in a 96-well plate format. The method can be used either quantitatively (qRT-PCR-based MethyLight) or qualitatively (using agarose gels) to detect changes in DNA methylation; both are described in this protocol.
November 16, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803282/preparing-polymerase-chain-reaction-pcr-products-for-capillary-sequencing
#7
Elaine Mardis, W Richard McCombie
This protocol describes the preparation of amplified products for use in Sanger-based capillary DNA sequencing, for example, to verify a clone or a construct. Amplified samples, subsequently treated with a mixture of exonuclease and shrimp alkaline phosphatase to remove unincorporated primers and dNTPs left from polymerase chain reaction (PCR), may be used directly for sequencing. This protocol relies on the use of the Biomek FX Workstation or a multichannel pipettor.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803281/agarose-gel-size-selection-for-dna-sequencing-libraries
#8
Elaine Mardis, W Richard McCombie
Agarose gel electrophoresis may be used to purify fragmented genomic DNA after ligation of adaptors. After electrophoresis, the region of the gel containing the desired size range of DNA is excised, and the DNA is subsequently extracted from the gel and purified by passage through a spin column.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803280/whole-genome-sequencing-manual-library-preparation
#9
Elaine Mardis, W Richard McCombie
This protocol describes a manual approach for the preparation of genomic DNA libraries suitable for Illumina sequencing. Genomic DNA fragments produced by shearing by sonication are ligated to adaptors and amplified by polymerase chain reaction (PCR). The amplified DNA, separated by size and gel-purified, is suitable for use as template in whole-genome sequencing.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803279/whole-genome-sequencing-automated-nonindexed-library-preparation
#10
Elaine Mardis, W Richard McCombie
This protocol describes an automated procedure for constructing a nonindexed Illumina DNA library and relies on the use of a CyBi-SELMA automated pipetting machine, the Covaris E210 shearing instrument, and the epMotion 5075. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Here, double-stranded DNA is fragmented when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymerase chain reaction (PCR), and subjected to size selection using magnetic beads...
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803278/preparing-plasmid-subclones-for-capillary-sequencing
#11
Elaine Mardis, W Richard McCombie
This protocol describes the preparation of plasmid DNA from bacterial cultures or from archived cultures, based on the standard miniprep method. The resulting DNA is suitable in quantity and quality for use as template in capillary DNA sequencing. The protocol relies on the use of the Biomek automated liquid handling system.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803277/library-quantification-using-picogreen-fluorometry
#12
Elaine Mardis, W Richard McCombie
This protocol describes the quantification of DNA using PicoGreen and a fluorometer to determine the concentration of DNA sample for downstream processing. Because dye-based methods will not detect degraded or short DNA fragments, PicoGreen requires DNAs ≥50 bp, but can less reliably detect fragments as small as 20 bp.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803276/automated-library-preparation-for-dna-sequencing
#13
Elaine Mardis, W Richard McCombie
This protocol describes a generalized automated procedure for constructing indexed and nonindexed Illumina DNA libraries.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803275/rna-seq-rna-conversion-to-cdna-and-amplification
#14
Elaine Mardis, W Richard McCombie
The Ovation RNA-Seq Kit provides a fast and simple method for preparing amplified cDNA from total RNA. Amplification is initiated both at the 3' ends of the transcripts and across the entire range of transcribed sequences; thus, this approach is ideal for next-generation sequencing, because the reads are distributed across the transcript types. The amplified cDNA produced in this protocol is used to create libraries optimized for use in the Illumina Genome Analyzer II platform. A procedure for removing small degraded RNAs before the sample is processed into cDNA is also included...
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803274/solution-phase-exome-capture
#15
Elaine Mardis, W Richard McCombie
This protocol describes the construction of a paired-end library of genomic DNA (gDNA) and subsequent capture of specific regions of a genome using NimbleGen sequence capture probes and Illumina TruSeq oligos. The captured DNA, purified and quantitated, is appropriate for use as template in Illumina sequencing systems. A procedure is also provided for magnetic AMPure bead cleanup.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803273/preparation-of-an-8-kb-mate-pair-library-for-illumina-sequencing
#16
Elaine Mardis, W Richard McCombie
The "mate-pair" approach for dual end sequencing uses long library fragments (1000-10,000 bp) that are circularized by ligation to the ends of a single, common DNA adaptor of known sequence. Once the ends are mated to the adaptor, a final library of fragments that retain the adaptor and only the ends of the original, circularized piece of DNA can be obtained. This protocol provides instructions for preparing an 8-kb paired-end library suitable for sequencing on the Illumina instrument. These are large-insert libraries, and the initial shearing will use various parameters and different instruments to achieve the desired high-size fraction...
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803272/preparation-of-a-3-kb-mate-pair-library-for-illumina-sequencing
#17
Elaine Mardis, W Richard McCombie
The "mate-pair" approach for dual end sequencing uses long library fragments (1000-10,000 bp) that are circularized by ligation to the ends of a single, common DNA adaptor of known sequence. Once the ends are mated to the adaptor, a final library of fragments that retain the adaptor and only the ends of the original, circularized piece of DNA can be obtained. This protocol describes how to prepare a 3-kb mate-paired-end library. The resulting amplified DNA is suitable for sequencing on the Illumina sequencer...
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803271/library-quantification-fluorometric-quantitation-of-double-stranded-or-single-stranded-dna-samples-using-the-qubit-system
#18
Elaine Mardis, W Richard McCombie
Qubit is an accurate and highly sensitive fluorescence-based quantitation system. Several assay kits have been optimized for the Qubit fluorometer, but also function efficiently with other fluorometers. The high-sensitivity dsDNA Qubit Kit has a detection range of 0.2-100 ng. The ssDNA Kit has a detection range of 1-200 ng.
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803270/whole-genome-sequencing-automated-indexed-library-preparation
#19
Elaine Mardis, W Richard McCombie
This protocol describes an automated procedure for constructing an indexed Illumina DNA library. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Double-stranded DNA (dsDNA) will fragment when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymer chain reaction (PCR), and subjected to size selection using magnetic beads. The product is suitable for use as template in whole-genome sequencing...
November 1, 2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27803269/cycle-sequencing-reactions
#20
Elaine Mardis, W Richard McCombie
Capillary sequencing of DNA remains a mainstay in the toolkit of molecular biologists. Whether the technique is used to verify that a clone is constructed properly or to validate an interesting variant found by next-generation whole-genome sequencing, capillary sequencing is an extremely versatile tool. This method for cycle sequencing relies on the use of the ABI3730xl capillary sequencer. The template can be prepared from plasmid minipreps for direct sequencing of clones to identify a mutation or structure of interest, or amplified products can be sequenced individually with each of the two primers used in LongAmp amplification reactions...
November 1, 2016: Cold Spring Harbor Protocols
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