Project description:Despite the precipitous decline in the cost of genome sequencing over the last few years, library preparation for RNA-seq is still laborious and expensive for high throughput screening for drug discovery. Limited availability of RNA generated by some experimental workflows poses an additional challenge and typically adds to the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that also maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies – Swift and Swift Rapid – using the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics, as our reference. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark differential gene expression in these kits, at input quantities ranging between 10 ng to 500 ng. Read quality and alignment metrics revealed high mapping efficiency and uniform read coverage through genes for all samples across all three kits. Normalized read counts between all treatment groups were in high agreement, with pairwise Pearson correlation coefficients >0.97. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits are cost effective and offer shorter workflow times enabled by their patented Adaptase technology. Furthermore, the Swift RNA kit allows for a relatively broader (and lower) input range, producing consistent results across diverse samples. The Swift Rapid RNA method is the fastest and most cost effective NGS workflow that is best suited for higher RNA yields, with the exact same RNA input range as the Illumina TruSeq kit. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways attributable to input mRNA concentration.
Project description:We evaluated the effect of the small RNA library preparation method on 5' tRNA-halves and miRNA abundance in libraries prepared from serum RNA using three commercially available small RNA library preparation kits (TruSeq small RNA library preparation kit v2 (Illumina), TailorMix miRNA sample preparation kit v2 (Seqmatic) and the NEBNext Multiplex Small RNA library prep kit (New England Biolabs)). RNA isolated from 100 µl of serum collected from healthy mice was used as input for the preparation of a small RNA library in duplicate and libraries were single end sequenced.
Project description:Total RNA, greater than 100ng, from cultured cells was isolated in TRIzol L and purified using Qiagen RNeasy Mini Kit per manufacturer’s protocols. Agilent Technologies 2100 Bioanalyzer was used to assess the RNA quality. RNA libraries were prepared and multiplexed using Illumina TruSeq RNA Library Preparation Kit v2 (non-stranded and poly-A selection) and 10 nM of cDNA was used as the input for high-throughput sequencing via Illumina’s HiSeq 2500 platform, producing 50 bp paired end reads.
Project description:YAV20 (E7946 loxP[dciA] SpR lacZ::cre ZeoR), AB14 (YAV20 inv[glmU-mioC]) and AB23 ( YAV20 ΔrecB::CmR) were created by natural transfromation using cognate plasmids. Cells were grown in the M( minimla media supplemented with fructose, with or without arabinose (ara). Genomic DNA was extracted with the Sigma GenElute® bacterial genomic DNA kit to generate a genomic library according to Illumina’s protocol. The libraries and the sequencing were performed by the High-throughput Sequencing facility of the I2BC (https://www.i2bc.paris-saclay.fr/sequencing/ng-sequencing/, CNRS, Gif-sur-Yvette, France). Genomic DNA libraries were made with the ‘Nextera DNA library preparation kit’ (Illumina) following the manufacturer’s recommendations.
Project description:Input mRNAs and ribosome protected fragments (RPFs) from wildtype and Mettl5 knockout (KO) B16 cells were analyzed by TruSeq Ribo Profile (Illumina) library preparation and high throughput sequencing
Project description:Input mRNAs and ribosome protected fragments (RPFs) from wildtype and Mettl5 knockout (KO) mESC cells were analyzed by TruSeq Ribo Profile (Illumina) library preparation and high throughput sequencing
Project description:Input mRNAs and ribosome protected fragments (RPFs) from Heterozygout (Control) and Dis3l2 knockout (KO) mESCs cultures were analyzed by TruSeq Ribo Profile (Illumina) library preparation and high throughput sequencing
Project description:Expression profiles of Saline, OVA, and Herb treated mice samples. RNA extracted with the Purelink RNA kit was used for cDNA library preparation with the TruSeq Library Prep Kit (Illumina) and sequenced on the NextSeq500.
Project description:In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5’ and 3’ end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.
Project description:In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5’ and 3’ end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.
