Unknown,Transcriptomics,Genomics,Proteomics

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Integrated genome and transcriptome sequencing from the same cell

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Lennart Kester

PROVIDER: E-GEOD-62952 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Anthocyanins are flavonoid compounds responsible for red/purple colours in the leaves, fruit and flowers of many plant species. They are produced through a multistep pathway which is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are non-functional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification and transport in Shiraz. One up-regulated gene ANTHOCYANIN 3- O-GLUCOSIDE-6â-O-ACYLTRANSFERASE (Vv3AT) encodes a BAHD acyltransferase protein, belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilise both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In V. vinifera cv. Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. Transgenic Chardonnay/Shiraz and non-transformed WT controls were all grown in the same glasshouse in ambient light with a night break. Day and night temperatures were approximately 27oC and 22oC respectively. For microarray experiments, whole berries were sampled from independent transgenic lines: three from transgenic Chardonnay and four from transgenic Shiraz, resulting in three and four biological replicates respectively. Bunches were harvested close to ripeness based on average total soluble solids (TSS, measured as oBrix). This was aimed to be between 20 â 24 oBrix and was determined from TTS of a subsamples from each bunch (Table S53). A sample consisted of all remaining berries from a single bunch except when there were <100 berries in which case more than one bunch was used in the one replicate. Whole berries were immediately frozen in liquid nitrogen. For skin samples, the skins were first removed from fresh berries then frozen in liquid nitrogen. All samples were stored at -80oC.

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Integrated genome and transcriptome sequencing from the same cell

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