Project description:The goal of the study was to compare gene expression of Robo1+/+ and Robo1-/- luminal progenitors. Total RNAs were then extracted from FACS purified luminal progenitor cells, harvested from Robo1+/+ or Robo1-/- mice (n=3 per genotype, two animals per n) using TRIreagent LS (Sigma, T3934). Poly(A)+ RNA sequencing libraries were made from each sample using the TruSeq RNA library preparation kit v.1 (Illumina). Illumina RNA PolyA library preparation guide. A total of 6 libraries were created by PCR amplification with Illumina barcoding primers using kit recommended conditions and quantified using a Bioanalyzer DNA 1000 kit (Agilent).
Project description:Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report an essential role for the germline-specific paralog of the mRNA cap-binding factor eIF4E, known as eIF4E1b, in the storage and repression of maternal mRNAs with short polyA tails. eIF4E1b binds to the mRNA cap and is targeted to ribonucleoprotein complexes through its direct interaction with eIF4ENIF1/4E-T. In early embryos, eIF4E1b binds to a specific set of translationally repressed mRNAs with short or no polyA tails, such as histone mRNAs, which are translated later on during embryogenesis. Consistent with an important role in maternal mRNA dormancy, mutation of eIF4E1b in zebrafish impairs female germline development. Understanding the mechanism and function of eIF4E1B provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.
Project description:Purpose: Profile global expression of prostatic tumors to compare with published datasets. Methods: Prostate tumors were flash frozen followed by processed for total RNA using the RNeasy kit (Qiagen). Samples were submitted for polyA library preparation and bulk RNA-sequencing. Conclusions: Global gene expression profiling and comparison of prostate tumor genotypes suggests differential enrichment of neuroendocrine signatures that match established human neuroendocrine prostate cancer expression signatures.
Project description:We perturbed the expression levels of circSlc45a4 and mRNA Slc45a4 in developing mouse cortex (E15.5) by RNAi/shRNAs. 48 h post-electroporation the cells were isolated by FACS (GFP+), RNA was extracted with Trizol and polyA+ libraries were generated with Illumina TruSeq Stranded mRNA LT Sample Prep Kit. We find many significant changes in the transcriptomic profile after knockdown of circSlc45a4 and mRNA Slc45a4, that are distinct from each other.
Project description:We perturbed the expression levels of circSLC45A4 in the human neuroblastoma cell line (SH-SY5Y) by RNAi/siRNAs. 96 h post-knockdown the cells were harvested, RNA extracted with Trizol and polyA+ libraries were generated with Illumina's TruSeq RNA Library Prep Kit v2. We find many significant changes in the transcriptomic profile after knockdown of circSLC45A4 and the induction of many genes connected to nervous system development, neuron projection development, synapse organization and cell adhesion.
Project description:The goal of the study was to compare gene expression of P0 wild-type, P0 Fezf2-/- cortices, and Fezf2-/-; Fezf2-EnR cortices. Total RNAs were isolated from P0 cortices dissected from wild-type (n=3), Fezf2-/- mice (n=4), and Fezf2-/-; Fezf2-EnR cortices (n=2), following Qiagen RNAeasy kit instruction.Sequence libraries were made following Illumina RNA PolyA library preparation guide.The libaries were pair-end sequenced (150nt per end). Differentially expressed genes were identified by DESEQ.
Project description:Valproate(VPA) has been used in the treatment of bipolar disorder since the 1990s. However, the therapeutic targetsof VPA have remained elusive. Here we used RNA sequencing in human iPSCs derived from bipolar patients to further identify important molecular targets. Human iPSCs were homogenized and total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany). RNA quantity and quality were assessed using fluorometry (Qubit RNA Broad Range Assay Kit and Fluorometer; Invitrogen, Carlsbad, CA) and chromatography (Bioanalyzer and RNA 6000 Nano Kit; Agilent, Santa Clara, CA), respectively. Libraries were prepared using TruSeq Stranded mRNA (PolyA+) kit (Illumina, San Diego, CA) and sequenced by Illumina NextSeq 500. The read length was 75bp with 30-40M reads per sample. FastQC (v0.11.3) was performed to assess data quality. TopHat2 (v2.0.9) aligned the reads to the mouse reference genome (Mus musculus UCSC mm10) and to the Ensembl human reference genome (GRCh38.p13) using default parameters. Alignments were then converted to expression count data using HTseq (v0.6.1) with default union mode.
Project description:Purpose: The aim of this study is to build up a transcriptomic data analysis pipeline and test its performance using synthetic "sequins" controls and lung cancer cell lines. Methods - Cell Culture: Lung adenocarcinoma cell lines NCI-H1975 and HCC827 from a range of passages (2-4) were grown on 3 separate occasions in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and 1% penicillin-streptomycin. Methods - RNA preparation: Total RNA was extracted using the TRIzol reagent. 6 ng of sequins were added to 1 ug of total RNA (mix A with H1975, mix B with HCC827). Methods - Library prep: the sequin+total RNA mixtures were subjected to polyA selection (NEBNext PolyA E7490) and library prep (NEBNext Ultra II Directional RNA Library Prep Kit for Illumina) following manufacturer's instructions; we performed 9 PCR cycles