Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research

Zhang, Meiping; Zhang, Yang; Scheuring, Chantel F.; Wu, Chun; Dong, Jennifer J.; Zhang, Hongbin

doi:10.1038/nprot.2011.455

Cited by 121 publications

(100 citation statements)

References 49 publications

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“…One million P51R cells were encapsulated in agarose plugs, DNA was extracted, restricted with PacI, and separated on a CHEF pulse field electrophoresis apparatus as described by Zang et al 26 Size markers were prepared by ligating l DNA digested with NheI, or Kas1. About 50 to 100 ng of size-purified genomic DNA was obtained.…”

Section: Reduced Representation Methyl-seqmentioning

confidence: 99%

Mechanisms of establishment and functional significance of DNA demethylation during erythroid differentiation

Bartholdy¹,

Lajugie²,

Yan³

et al. 2018

Blood Advances

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Key Points• We have generated allele-specific base resolution methylomes of primary basophilic erythroblasts.• DNA demethylation during differentiation of HSPC into BasoE occurs mostly in inactive regions causing formation of PMD in 74% of methylome.Erythroid differentiation is associated with global DNA demethylation, but a complete methylome was lacking in the erythroid lineage. We have generated allele-specific base resolution methylomes of primary basophilic erythroblasts (BasoEs) and compared these with 8 other cell types. We found that DNA demethylation during differentiation from hematopoietic stem/progenitor cells (HSPCs) to BasoEs occurred predominantly in intergenic sequences and in inactive gene bodies causing the formation of partially methylated domains (PMDs) in 74% of the BasoE methylome. Moreover, differentially methylated regions (DMRs) between HSPCs and BasoEs occurred mostly in putative enhancer regions and were most often associated with GATA, EKLF, and AP1 binding motifs. Surprisingly, promoters silent in both HSPCs and BasoEs exhibited much more dramatic chromatin changes during differentiation than activated promoters. Unmethylated silent promoters were often associated with active chromatin states in highly methylated domains (HMDs) but with polycomb-repression in PMDs, indicating that silent promoters are generally regulated differently in HMDs and PMDs. We show that long PMDs replicate late, but that short PMDs replicate early and therefore that the partial methylation of DNA after replication during erythroid expansion occurs throughout S phase of the cell cycle. We propose that baseline maintenance methylation following replication decreases during erythroid differentiation resulting in PMD formation and that the presence of HMDs in the BasoE methylome results from transcription-associated DNA methylation of gene bodies. We detected ;700 large allele-specific DMRs that were enriched in single-nucleotide polymorphisms, suggesting that primary DNA sequence might be a determinant of DNA methylation levels within PMDs.

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Section: Reduced Representation Methyl-seqmentioning

confidence: 99%

Mechanisms of establishment and functional significance of DNA demethylation during erythroid differentiation

Bartholdy¹,

Lajugie²,

Yan³

et al. 2018

Blood Advances

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“…Our method uses an updated version of the 10x microfluidic gel bead partitioning system (Zheng et al 2016), called the Chromium Genome Reagent Kit (Methods). Library construction starts from 1.25 ng of DNA having size 50 kb or longer (Zhang et al 2012), from a single individual organism or clonal population (such as a cell line).…”

Section: Data Generationmentioning

confidence: 99%

Direct determination of diploid genome sequences

et al. 2017

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Determining the genome sequence of an organism is challenging, yet fundamental to understanding its biology. Over the past decade, thousands of human genomes have been sequenced, contributing deeply to biomedical research. In the vast majority of cases, these have been analyzed by aligning sequence reads to a single reference genome, biasing the resulting analyses, and in general, failing to capture sequences novel to a given genome. Some de novo assemblies have been constructed free of reference bias, but nearly all were constructed by merging homologous loci into single "consensus" sequences, generally absent from nature. These assemblies do not correctly represent the diploid biology of an individual. In exactly two cases, true diploid de novo assemblies have been made, at great expense. One was generated using Sanger sequencing, and one using thousands of clone pools. Here, we demonstrate a straightforward and low-cost method for creating true diploid de novo assemblies. We make a single library from ∼1 ng of high molecular weight DNA, using the 10x Genomics microfluidic platform to partition the genome. We applied this technique to seven human samples, generating low-cost HiSeq X data, then assembled these using a new "pushbutton" algorithm, Supernova. Each computation took 2 d on a single server. Each yielded contigs longer than 100 kb, phase blocks longer than 2.5 Mb, and scaffolds longer than 15 Mb. Our method provides a scalable capability for determining the actual diploid genome sequence in a sample, opening the door to new approaches in genomic biology and medicine.

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“…Isolated nuclei can also be cleaned extensively to remove contaminants that are generally in the cytoplasm [39]. [ 1 8 _ T D $ D I F F ] Contaminants are removed through use of a strong reducing agent (e.g., 2-mercaptoethanol), detergents (e.g., Triton-X 100), and polymers [e.g., polyvinylpyrrolidone (PVP)] that prevent the oxidation of polyphenols, solubilize lipids and enzymes, and bind polyphenols, all of which can reduce DNA quality [38][39][40][41]. Furthermore, a Percoll density gradient is used to help separate nuclei from particles of a different density [42].…”

Section: Data Collectionmentioning

confidence: 99%