Methods for analyzing RNA expression and DNA replication from single cells

Abstract

To provide single-cell multi-omics with data quality comparable to that obtained when DNA replication analysis and whole transcriptome analysis are performed on different single cells. [Solution] A method for analyzing RNA expression and DNA replication from a single cell, comprising: (1) a step of separating genomic DNA and RNA from a cell population, one cell at a time; (2) a step of performing whole-genome amplification on the genomic DNA separated in step (1) for each cell, and performing whole-genome analysis of the amplified genomic DNA to evaluate the total DNA copy number of the genome; (3) a step of preparing a cDNA library from each cell by whole-transcriptome amplification using the RNA separated in step (1) as a template, and performing gene expression analysis to obtain gene expression data, wherein the whole-transcriptome amplification includes introducing a nick into the cDNA using the nuclease activity of a DNA strand-specific RNA:DNA hybrid strand degrading enzyme during the reverse transcription reaction by RNase H-minus reverse transcriptase, and using a not-so-random (NSR) primer; and (4) a step of analyzing the correlation between DNA replication and gene expression at the genome-wide single-cell level.

Claims

[Claim 1] A method for analyzing RNA expression and DNA replication from a single cell, (1) For a cell population, the process of separating genomic DNA and RNA from each individual cell, (2) For each cell, whole-genome amplification is performed on the genomic DNA separated in step (1), and whole-genome analysis is performed on the amplified genomic DNA to evaluate the total DNA copy number of the genome. (3) A step to prepare a cDNA library by whole transcriptome amplification using the RNA separated in step (1) as a template for each cell, and to obtain gene expression data by performing gene expression analysis, wherein the whole transcriptome amplification includes introducing a nick into the cDNA using the nuclease activity of a DNA strand-specific RNA:DNA hybrid strand degrading enzyme during the reverse transcription reaction by RNase H-minus reverse transcriptase, and using a not-so-random (NSR) primer, and (4) A process for analyzing the correlation between DNA replication and gene expression at the genome-wide single-cell level. Methods that include... [Claim 2] Step (4) is (a) A step of determining the genome replication rate of cells in a population of S-phase cells based on the total DNA copy number of the genome, sorting the cells according to the genome replication rate, and detecting genes whose expression changes during the progression of S-phase. (b) Determining replication timing (RT) profiles based on the total DNA copy number of the genome, and detecting replication timing asynchronous and / or bias in gene expression ratios between alleles and / or haplotypes, or (c) A process to detect copy number variation (CNV) in a non-S phase cell population and to analyze the gene expression levels associated with CNV. The method according to claim 1. [Claim 3] The method according to claim 1, wherein genomic DNA is separated in step (1) by binding chromatin to a solid support having carboxylic acid group modification. [Claim 4] The method according to claim 1, wherein the RNA isolated in step (1) includes both poly(A)RNA and non-poly(A)RNA. [Claim 5] The method according to claim 1, wherein the whole genome amplification in step (2) is based on degenerate oligonucleotide-primed PCR technology. [Claim 6] The method according to claim 2, wherein the cellular genome replication rate is determined by mapping reads to a reference genome and replicating or binarizing each genomic bin based on the total DNA copy number of the genome. [Claim 7] The method according to claim 1, wherein the NSR primer is a primer obtained by removing a sequence that recognizes ribosomal RNA (rRNA) from a random primer. [Claim 8] The method according to claim 1, which can be used for analyzing genomic heterogeneity in a cell population of cancer tissue or for prenatal chromosomal diagnosis. [Claim 9] The method according to claim 8, wherein the expression of imprinted genes is analyzed in prenatal chromosomal diagnosis.

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