Project description:Measurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from mature tRNA with a simple-to-use computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in multiple eukaryotes and is applicable to any organism with a known genome.
Project description:Current next-generation RNA sequencing methods do not provide accurate quantification of small RNAs within a sample due to sequence-dependent biases in capture, ligation, and amplification during library preparation. We present a method – AQRNA-seq – that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for small RNAs in a sample. The library preparation and data processing steps were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying lengths, and northern blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied as a function of cancer progression, while application to bacterial tRNA pools, a traditionally hard-to-sequence class of RNAs, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced tRNA-driven codon-biased translation. AQRNA-seq thus provides a means to quantitatively map the small RNA landscape in cells.
Project description:Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to detect mistranslation from two different tRNA variants. The first variant, tRNA-Pro-G3:U70, has a mutation in its acceptor stem creating a G3:U70 base pair which is the key identity element for the alanine tRNA synthetase. This tRNA should be charged with alanine and mis-incorporate alanine at proline codons. The second variant, tRNA-Ser-UCU,G26A, is a serine tRNA with an arginine anticodon and a G26A secondary mutation to dampen function and prevent lethal levels of mistranslation. This tRNA should mis-incorporate serine at arginine codons.
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:Deep sequencing of tRNAs have historically been nutoriously difficult. Here, we benchmark a newly developed library prep protocol termed OTTR agains intact tRNAs as well as tRNA fragments and show that OTTR outperforms any other commercial cloning protocol.
Project description:We created a comprehensive tRNA deletion library in yeast and characterized the phenotypic and further characterized the molecular changes in a subset of deletion strains Each tRNA deletion strain was created using homologous recombination by introduction of an antibiotic cassette. Each strain was grown at 30°C in rich medium to mid log phase from which mRNA was extracted
Project description:CAMPARI2 CRISPR screening for SOCE modulators of ER stress. PC cells were sorted and sequenced for CRISPR whole KO library (De brie). Unsorted, SOrte din LOW PC and LOW PC Tunica treated fro 4hours were analysed.
Project description:We created a comprehensive tRNA deletion library in yeast and characterized the phenotypic and further characterized the molecular changes in a subset of deletion strains
Project description:In this study we subjected a library of synthetic sRNAs to multiple rounds of ethanol selection and analyzed changes in sRNA variant abundance by high-throughput sequencing.
Project description:We developed a Ligation-based Library vs Library high-throughput Yeast Two-Hybrid (LLL-Y2H) screening system to explore protein interactions.
