An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments
ABSTRACT: Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs, variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved. Deep sequencing libraries from either a randomized RNA oligo or an equimolar miRNA mix were analyzed for evenness of capture.
Project description:Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs, variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved. Overall design: Deep sequencing libraries from either a randomized RNA oligo or an equimolar miRNA mix were analyzed for evenness of capture.
Project description:Removal of introns by pre-mRNA splicing is a critical and in some cases rate-limiting step in mammalian gene expression. Deep sequencing of mouse embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within poly(A) selected transcripts; we classify these as “detained” introns (DIs). We identified thousands of DIs flanking both constitutive and alternatively spliced exons in human and mouse cell lines. Drug inhibition of Clk SR-protein kinase activity triggered rapid splicing changes in a specific set of DIs, about half of which showed increased splicing and half increased intron detention, altering the transcript pool of over 300 genes. These data suggest a widespread mechanism by which a nuclear detained pool of mostly processed pre-mRNAs can be rapidly mobilized in response to stress or homeostatic autoregulation. v6.5 mouse embryonic stem cells were untreated, treated with the Clk kinase inhibitor KH-CB19, or treated with DMSO as a negative control. Untreated cells were harvested and a single replicate was sequenced using a custom, ligation-based, stranded library preparation protocol. Treated cells were harvested at time 0 and at 2 hours post-treatment, and poly(A)-selected RNA-seq libraries were made from biological duplicates for each treatment/time, barcoded, and sequenced by strand-specific, paired-end sequencing using the Illumina TruSeq kit.
Project description:To test whether the RNA Polymerase II factor PAF1 affects PIWI silencing, we examined the transcriptome expression levels in Drosophila OSS cells following siRNA knockdown of these factors. Overall design: The total RNAs from OSS cells were extracted after siRNA knockdown of GFP negative control, PAF1, PIWI, and a double knockdown of both PAF1 and PIWI. The RNAs were then converted into strand-specific sequencing libraries with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina.
Project description:Deep sequencing of transcriptomes allows quantitative and qualitative analysis of many RNA species in a sample, with parallel comparison of expression levels, splicing variants, natural antisense transcripts, RNA editing and transcriptional start and stop sites the ideal goal. By computational modeling, we show how libraries of multiple insert sizes combined with strand-specific, paired-end (SS-PE) sequencing can increase the information gained on alternative splicing, especially in higher eukaryotes. Despite the benefits of gaining SS-PE data with paired ends of varying distance, the standard Illumina protocol allows only non-strand-specific, paired-end sequencing with a single insert size. Here, we modify the Illumina RNA ligation protocol to allow SS-PE sequencing by using a custom pre-adenylated 3’ adaptor. We generate parallel libraries with differing insert sizes to aid deconvolution of alternative splicing events and to characterize the extent and distribution of natural antisense transcription in C. elegans. Despite stringent requirements for detection of alternative splicing, our data increases the number of intron retention and exon skipping events annotated in the Wormbase genome annotations by 127 % and 121 %, respectively. We show that parallel libraries with a range of insert sizes increase transcriptomic information gained by sequencing and that by current established benchmarks our protocol gives competitive results with respect to library quality. Sequencing of mRNA from C. elegans with libraries of four differing insert sizes
Project description:Small RNAs, including microRNAs and their targets, as well as phased secondary siRNAs, were characterized in the soybean genome by deep sequencing of small RNA libraries from a wide range of tissues. The mRNA targets of many of these small RNAs were also validated from many of the same tissues using PARE (Parallel Analysis of RNA Ends) libraries. Overall design: Small RNA profiling was done by Illumina TruSeq sample preparation followed by high-throughput sequencing using an Illumina HiSeq 2500 at the Delaware Biotechnology Institute. PARE analysis used library construction approaches described in the journal Methods (PubMed ID: 23810899).
Project description:MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding ribonucleic acids (RNAs) that regulate gene expression by binding to their complementary sequences. Recent years, a great deal of miRNAs which highly-enriched in skeletal muscle have been identified, which can influence multiple facets of muscle development and function through their regulation of key genes controlling myogenesis. However, to date no miRNAs have been reported to modulate muscle development in goat. Total RNAs from the xuhuai goats longissimus thoracis at fetal and six month old stages were used to construct small RNA libraries for Solexa SBS technology sequencing. In the small RNA profile, a total of 15,627,457 clean reads were obtained from the fetal goat library and 15,593,721 clean reads from the six month old goat library. There are 471 conserved miRNAs overlapped in both libraries, of which 343 miRNAs were differential expressed. We identified 122 novel miRNAs in the fetal caprine library and 53 novel miRNAs in the six month old-caprine library. Overall design: Examination of Chinese Xuhuai goat miRNAs by deep sequencing
Project description:Strand-specific massively-parallel cDNA sequencing (RNA-Seq) is a powerful tool for novel transcript discovery, genome annotation, and expression profiling. Despite multiple published methods for strand-specific RNA-Seq, no consensus exists as to how to choose between them. Here, we developed a comprehensive computational pipeline for the comparison of library quality metrics from any RNA-Seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library construction protocols, including both published and our own novel methods. We found marked differences in complexity, strand-specificity, evenness and continuity of coverage, agreement with known annotations, and accuracy for expression profiling. Weighing each method’s performance and ease, we identify the dUTP second strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in any organism. Examination of 11 different strand-specific RNA-Seq libraries from 7 distinct methods; also 2 control non-strand-specific RNA-Seq libraries. To assess the performance of each strand-specific library in digital expression profiling, we compared them to reference expression measurements estimated from expression profiles using competitive hybridization of a mid-log RNA sample vs. genomic DNA using Agilent arrays.
Project description:To identify methylated small RNAs in C. elegans, we deep sequenced both β-eliminated and untreated small RNAs isolated from wild type C. elegans. Small RNAs were isolated from larval and adult stage C. elegans and either subjected to β-elimination or no treatment. Small RNA cDNA libraries were sequenced on an Illumina HiSeq instrument, and enrichment or depletion of small RNAs by β-elimination was assessed after library size normalization based on the number of mappable reads in each library.