The ligation of the adaptors to the first-strand cDNA is carried out under an optimized condition, minimizing the GC content bias. The adaptor-ligated cDNAs are size-selected using solid phase reversible immobilization beads 30 , treated with USER to degrade the deoxyuridine-containing non-ligating strands of the adaptors, PCR-amplified and subjected to massively parallel sequencing.
We did not pursue these options because these techniques have a few limitations, including low library yields see Supplementary Note 1. The libraries were prepared using wild-type WT mouse embryonic stem mES cells and Kdm1a -deficient mES cells 33 in biological duplicates. For the dUTP libraries, we followed the published protocol Fig. The final yield of a library preparation method is an important indicator of its utility, especially when RNA is available only in small amounts.
Multiplexed libraries were subjected to either single- or paired-end sequencing on an Illumina HiSeq instrument. After standard demultiplexing and filtering, the reads were mapped to the mouse transcriptome and genome using TOPHAT 34 , allowing up to two mismatches.
We first noted that the percentage of reads that mapped to the genome Table 1 , column: alignment rate, total was higher for the DLAF libraries than the dUTP libraries. Higher mappability was consistent in WT Next, we determined the percentage of reads that mapped to unique regions of the annotated genome Table 1 , column: alignment rate, unique.
Interestingly, the percentages of such reads were substantially lower in Kdm1a -deficient mES cells than in libraries from WT mES cells, regardless of the methods used. The decrease in the percentages of the unique reads in Kdm1a -deficient mES cells could have been a result of an increased expression of murine endogenous retrovirus MuERV-L or other retrotransposable elements upon the loss of Kdm1a Taken together, the DLAF libraries showed a consistently higher mappability to non-repetitive genic regions than did the dUTP libraries.
Next, we compared the coverage along the length of the genes. Using RNA-SeQC 28 , 35 , the genes were first categorized into top-, middle- and bottom-expressed groups based on their expression levels. No significant difference was noted in the middle of the transcripts. Relative coverage for each percentile of gene length for the 5, middle-expressed genes in each library. Data are shown for individual replicates dashed lines and averaged replicates solid line from WT mES cells.
RNA-SeQC coverage is shown normalized to the total number of reads mapping to the 5, middle-expressed genes. Promoter antisense transcripts have been captured only after an enrichment of unstable RNA species 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , such as nascent RNA or small RNAs.
Reads aligning to the antisense strand are plotted on the negative y axis. For example, one CAGE study 12 revealed distinct groups of genes based on the modes of their promoter usages.
As shown in Fig. For a BP class gene, such as Ywhae Fig. We reason that mature miRNAs were lost during the size-selection step of the library preparation. Green: miRNA-Seq signal. Such peaks were not detected by the dUTP method. Coverage is shown as per gene per million non-rRNA reads. Data for individual replicates are shown as thin lines.
We postulated that computational trimming of poly A tails would enable these unmapped reads to align to the genome. Then, we trimmed T 9 and mapped them to the assembly again. Similar trends were observed for both top- and bottom-expressed genes in the WT and Kdm1a -deficient mES cells Supplementary Figs 7 and 8. Data are shown for Average of two biological replicates is shown and error-bars indicate the range of data.
The increased full-length coverage could be visualized in a genome browser at many individual loci Supplementary Fig. In the previous comparative study, the dUTP method outperformed many other methods in terms of these criteria As shown in Supplementary Fig.
Between the independent replicates, the DLAF and dUTP libraries showed equally high reproducibility, which was further confirmed by a lower coefficient of variation of gene expression, as calculated by Cuffdiff and CummeRbund 48 Supplementary Fig. The DLAF libraries showed low average variations in evenness of coverage, which were slightly but significantly higher than those of the dUTP libraries 5. The complexities of the libraries were calculated as fractions of reads with unique starting positions The sources of these improvements in strand-specificity and complexity are unclear.
In summary, apart from a slightly lower evenness of coverage, the DLAF libraries exhibited higher overall quality across multiple performance metrics. ScriptSeq libraries showed a significantly higher overall mapping rate and higher strand-specificity see Supplementary Note 5. However, the ScriptSeq libraries showed lower library yields and a lower reproducibility. In addition, ScriptSeq libraries displayed significantly higher gap percentages and lower evenness of coverage, regardless of the expression levels, indicating a highly discontinuous coverage of transcripts see Supplementary Note 5 for a possible explanation.
These data demonstrate that, although the ScriptSeq had a higher mapping rate than did the DLAF, the mapped ScriptSeq reads represent lower reproducibility and a biased population of the transcripts. Thus, lower library yield and more discontinuous and uneven coverage by the ScriptSeq libraries could be attributed to preferential hybridization of ScriptSeq oligonucleotides to RNA species that contain sequences complementary to the tagging sequence.
Data are averaged from three biological replicates. The sequence shows a clear bias towards GATCT, which is similar to a part of template-switching oligo.
The coverage is normalized to the total number of reads mapping to the 5, middle-expressed genes in each library. The first bases are plotted across the TSSs. In b and c , dashed and solid lines denote individual and averaged replicates obtained from 5, middle-expressed genes. We then calculated the coverage across each percentile of their lengths. This coverage pattern was noticeably improved compared with that of the dUTP libraries, which began declining at the tenth percentile Fig.
RNA-sequencing libraries prepared using many of the current methods, including the dUTP method, show an underrepresentation of one or both transcript ends In this study, we developed a novel and relatively simple method, the DLAF, for preparing libraries for ssRNA-seq with markedly high coverage at both ends of transcripts. Our results indicate a versatile utility of DLAF for gene expression analysis and mechanistic study of gene regulation.
In contrast, in the middle of transcripts, RT can initiate or terminate at any position because RNA is randomly fragmented Supplementary Fig. The exact genomic position where the transcription of a gene starts is a critical piece of information in the study of mechanisms that control actions of RNA polymerases, such as recruitment, pausing and initiation.
Meanwhile, the use of alternative polyadenylation sites is a prevalent mechanism of mRNA regulation in organisms from yeasts to mammals To map poly A -containing reads, several strategies have been employed, such as the use of a seed sequence or loosening the mapping stringency 24 , mapping reads to a transcripts database 18 and computationally removing the poly T tails In addition, these methods do not allow us to profile non-polyadenylated genes, such as mammalian histone genes, which can be readily detected in DLAF libraries Supplementary Fig.
Single-cell RNA-seq has emerged as a landmark approach to understand the behaviour of individual cells instead of whole populations 51 , 52 , 53 , However, the current methods for single-cell RNA-seq cannot preserve the strand information. Improving yields, strand specificity and the quality of libraries will be an important step towards a genuine single-cell transcriptome. A high library yield and a relatively short experimental workflow of DLAF might be suitable for meeting the demands for analysing multiple single-cell libraries.
However, further investigations are necessary to determine whether DLAF can be used to improve the transcriptome profiling of single cells. Tissue culture dishes were coated with 0. Cortices from E Cortices were incubated with 0. Half of culture media was replaced with new media every 5 days in vitro , and cells were harvested on 10 days in vitro.
All reagents for cell culture were from Life Technologies unless mentioned otherwise. The temperature of the reactions was increased slowly in a stepwise manner to avoid the dissociation of random primers from RNA molecules. To prepare the adaptors for ligation, six oligonucleotides with the sequences shown below were designed. They were then column purified. The ligated samples were size-selected using 1.
In general, we followed the initial protocol for dUTP library preparation 8 , with minor modifications. The original oligonucleotide sequence 8 was modified to maintain the orientation of sequencing, similar to that of the DLAF libraries. The cortices were dissected out from E After phase-separation, the supernatant was purified through Qiagen Mini Column.
The DNase I treatment and other purification steps were as described above. Treatment with RNases and other purification steps were as described above. They were then column purified and size-selected using 1. Two per cent of the library products were analysed by qPCR as described above. The same reactions were also amplified for 21 cycles in a conventional thermal cycler, and one-third volumes were analysed using polyacrylamide gel electrophoresis.
The mES libraries were subjected to both single-read and paired-end sequencing. Coverage across percentiles of gene length, coverage of intragenic and intergenic regions, coverage of gene ends, evenness of coverage and continuity of coverage were calculated using RNA-SeQC The data were normalized with total non-ribosomal and non-mitochondrial RNA reads. The coefficient of variation of gene expression was calculated using Cuffdiff v2.
The complexity of the libraries was estimated as the fraction of Multiplexed libraries were sequenced from one end for 50 bases by an Illumina Hiseq instrument. Only reads with shorter than 36 base inserts were mapped uniquely to mm9 assembly using bowtie v0. How to cite this article: Agarwal, S. Sequencing of first-strand cDNA library reveals full-length transcriptomes. See Supplementary Table 2 for sample and sequencing run details. Construction and characterization of a cDNA expression library from the endangered Hu sheep.
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Complementary techniques: Validation of gene expression data by quantitative real time PCR. Adv Exp Med Biol, Reverse Transcriptase. Schultz, S. Virus Research, Sterling, C. An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.
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Proceedings of the National Academy of Sciences,85 6 , Weinberg, E. Wu, N. Biological Procedures Online,12 1 , The Use of Reverse Transcriptase in Molecular Biology The mechanism behind reverse transcription has expanded the world of molecular biology by helping scientists overcome earlier obstacles by allowing scientists to use RNA as starting material instead of DNA. Choosing a Reverse Transcriptase for Your Experiment Different reverse transcriptases are suited for different situations.
One-step PCR processes have many advantages. They give consistent results, the protocol is simple and fast, and there is less pipetting.
It also does not provide stock cDNA. Because random primers are used, the two-step method often runs more efficiently. And it provides stock cDNA that allows for aliquoting and use in multiple assays.
The two step method does take much longer to perform and involves significantly more pipetting. A cDNA library will not. A genomic library will only offer us information on gene representation as they occur in the chromosome. It is similar in many ways to colony hybridization; however, this technique relies on the peptide sequence rather than the DNA sequence.
From there, the rest of the technique follows colony hybridization. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer.
In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Gene expression studies by quantitative reverse-transcription PCR qRT-PCR can be challenging when attempted with samples of a few cells or a single cell.
Although qRT-PCR studies provide valuable information about the relative expression of transcripts in a cell, they are difficult to perform with very small numbers of cells for example, 1—1, cells , for a variety of reasons.
These include difficulty in purifying extremely small amounts of total RNA, a lack of sensitivity, the need to collect samples often because of the very limited amount of RNA available and the inability to archive samples for future analysis. In some cases, we used a commercial cDNA-from-cells—type kit. The cDNA-from-cells and the no-RT control produced only nonspecific signals, as determined by melting curve analysis Fig.
The melting temperature of the nonspecific products was substantially different from that of the LDHA -specific amplicon. All experiments except the no-RT control were performed in duplicate. The horizontal orange line is the fluorescence threshold. T , temperature; RFU, relative fluorescence units.
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