Ribosome profiling of budding yeast Saccharomyces cerevisiae in eEF3 depleted conditions
ABSTRACT: Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of eEF3 depletion in yeast (Saccharomyces cerevisiae). eEF3 depletion was induced by methionine in a modified strain where the native promoter was replaced by methionine repressible MET25 promoter. Conditional depletion enables us to study global effects of an essential gene.
Project description:New1 is not an essential gene but its deletion shows a cold-sensitive phenotype in yeast Saccharomyces cerevisiae. In this study, we compare the NEW1 knockout effect on translation using Ribo-Seq and RNA-Seq analyses.
Project description:Loss of New1 leads to a cold-sensitive phenotype of yeast Saccharomyces cerevisiae. In this study we investigated the effect of NEW1 knockout on translation using Ribo-Seq and RNA-Seq analyses.
Project description:Based on the association of TP53 mutation and upregulated TP63 expression in the squamous subtype, we used cell lines derived from genetically engineered mouse models of PDAC (KRAS Trp53fl/+ and KRAS Trp53fl/+ Trp63fl/fl) to begin to unravel the functional consequences of these events in defining squamous tumours. RNA-Seq libraries were generated using TruSeq Stranded Total RNA (Part no. 15031048 Rev. D April 2013) kits, using on a Perkin ElmeraTMs Sciclone G3 NGS Workstation (Product no. SG3-31020-0300). Ribosomal depletion step was performed on 1 ug of total RNA using Ribo-Zero Gold prior to a heat fragmentation step aimed at producing libraries with an insert size between 120-200 bp. cDNA was then synthesized from the enriched and fragmented RNA using InvitrogenaTMs SuperScript II Reverse Transcriptase (Catalog no. 18064) and random primers. The resulting cDNA was further converted into double stranded DNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintain the strandedness of the library. Following 3aTM adenylation and adaptor ligation libraries were subjected to 15 cycles of PCR to produce RNA-Seq libraries ready for sequencing. Prior to sequencing, exome and RNA-Seq libraries were qualified and quantified via CaliperaTMs LabChip GX (Part no. 122000) instrument using the DNA High Sensitivity Reagent kit (Product no. CLS760672). Quantification of libraries for clustering was performed using the KAPA Library Quantification Kits For Illumina sequencing platforms (Kit code KK4824) in combination with Life Technologies Viia 7 real time PCR instrument. All libraries were sequenced using the Illumina HiSeq 2000/2500 system with TruSeq SBS Kit v3 - HS.
Project description:To define what genes are predominantly or specifically expressed in either soma or germline in C. elegans adults, total RNA was extracted from germline-less glp-4 mutant animals or from dissected gonads, respectively. Total RNA sequencing was peformed in duplicates. Four samples in total.
Project description:RNA sequencing (RNAseq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNAseq analysis. We have developed a set of spike-in RNA standards, termed ‘sequins’ (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and provides scaling factors for normalization between samples. We demonstrate the use of sequins in RNAseq experiments to measure sample-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA samples. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome. Detailed transcriptomic analysis of a human cell-type with synthetic RNA spike-ins ('sequins'). Sequins were initially combined at equimolar concentrations (a "flat" mix) and sequenced neat (i.e. without any natural RNA added). We then prepared two staggered mixtures (Mix A & B) and sequenced them neat. Mix A was then spiked into total RNA extracted from the K562 cell-type. Finally, we prepared a staggered mixture of fusion sequins and sequenced it neat.
Project description:Bone marrow (BM) cells were obtained by flushing the long bones of 8-week old C57BL/6 mice. BM cells were then were plated in macrophage SFM medium (Life Technologies) supplemented with penicillin-streptomycin and CSF-1 (Peprotech, 100 ng/ml) and cultured for one week to allow macrophage differentiation. BMDMs were polarized by adding IL-4 to the medium (40 ng/ml, Peprotech) for 72h or left untreated. 8 samples total, 2 groups, 4 biological replicates in each group
Project description:RNA-seq for monitoring expression levels in mutants that do not anchor chromatin at the nuclear periphery. RNA-seq of depleted rRNA samples of early embryo extracts for three different genotypes: wild-type, cec-4_delta and met-2 set-25_delta_delta, in two independent biological replicas
Project description:We carried out total RNA sequencing on RNA extracted from 5 brain subregions (cerebellum, cortex, hippocampus, hypothalamus and thalamus) and liver tissues of wild-type mice at 3 months of age. In total, two female and two male mice were used to obtain biological replicates for appropriate statistical analysis. Not only is this dataset used to perform expression analysis between brain subregions and between brain and liver, it is also used in-conjunction with 5hmC-seq methylation profiles that we performed to study the association between 5hmC and gene expression. Stranded RNA-seq library was prepared and sequenced on a HiSeq2500 by single end sequencing with 100 bp read length.