Project description:The genomic DNA of rearranged chromosome VIII (~900kb) of EC9 diploid strain was extracted from the gel of pulsed field gel electrophoresis (PFGE), and analyzed by array-CGH to identify it's amplified regions After PFGE, The ChrVIII DNA was purified from agarose gel and purified by column. Then the DNA was amplified by GenomePlex Whole Genome Amplification Kit for Array-comparative genomic hybridization.
Project description:The genomic DNA of wild-type chromosome VIII of evolved EC9-7 cells was extracted from the gel of pulsed field gel electrophoresis (PFGE), and analyzed by array-CGH to identify its chromosomal composition Yeast genomic DNA was extracted using QIAGEN Genomic-tip 100/G kit. After PFGE, The ChrVIII DNA was purified from agarose gel and purified by column. Then the DNA was amplified by GenomePlex Whole Genome Amplification Kit.
Project description:Single-cell human genome analysis using whole-genome amplified product is hampered by allele bias during amplification. Using an oligonucleotide SNP array, we examined the nature of the allele bias and its effect on the chromosomal copy number analysis. Keywords: single cell, copy number analysis, whole genome amplification, brain
Project description:Single-cell genomics and single-cell transcriptomics have recently emerged as powerful tools to study the biology of single cells at a genome-wide scale. Here we describe a method that allows the integration of genomic DNA and mRNA sequencing from the same cell. We use this method to correlate DNA copy number variation to transcriptome variability among individual cells. First, hand-picked single cells are lysed and reverse transcribed using a poly-A primer including cell-specific barcodes, a 5' Illumina adapter and a T7 promoter overhang to convert mRNA to single stranded cDNA (ss cDNA). The gDNA and single stranded cDNA are then subjected to quasilinear whole genome amplification, as previously described, using an adapter with a defined 27 nucleotide sequence at the 5M-bM-^@M-^Y end followed by 8 random nucleotides. After 7 rounds of amplification, the gDNA and cDNA are copied to generate a variety of different short amplicon (0.5M-bM-^@M-^S2.5 kb) species, with a majority of amplicons containing adapter Ad-2 at both ends and a small fraction of cDNA derived amplicons containing Ad-2 at one end and Ad-1x at the other. Next, the sample is split into two tubes to further amplify gDNA and cDNA. The tube used to sequence gDNA is amplified using PCR. Following sonication, adapter Ad-2 removal, and cell-specific indexed Illumina library preparation, this half is used to sequence gDNA. The tube used to sequence cDNA is converted to double-stranded cDNA and amplified using in vitro transcription such that the amplified RNA (aRNA) is uniquely produced from cDNA but not gDNA. 3M-bM-^@M-^Y Illumina adapters are then ligated to the aRNA followed by reverse transcription and PCR, allowing quantification of mRNA.
Project description:Single-cell human genome analysis using whole-genome amplified product is hampered by allele bias during amplification. Using an oligonucleotide SNP array, we examined the nature of the allele bias and its effect on the chromosomal copy number analysis. Experiment Overall Design: TB106, lymphoblastoid cell line; CMK11-5 and CMK86, cell line; WGA, whole-genome amplified from bulk DNA; SC, whole-genome amplified product from single cell. Genotype and copy number status were compared between non-amplified products and their single cell products.