Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.
Project description:We performed a genome-scale screen for suppressors of interferon stimulated gene (ISG) expression in human haploid cells (HAP1). Ubiquitin specific peptidase 14 (USP14) was a significant hit. In order to validate USP14 as a regulator of ISG expression, we created knockouts of USP14 in HAP1 cells using CRISPR-Cas9 and performed RNA-seq on coding RNA from USP14 KO and WT cells. This data was used to determine if ISGs were upregulated in USP14 KO HAP1 cells.
Project description:We performed a genome-scale screen for suppressors of interferon stimulated gene (ISG) expression in human haploid cells (HAP1). DEAD-box helicase 6 (DDX6) was a significant hit. In order to validate DDX6 as a regulator of ISG expression, we created knockouts of DDX6 in HAP1 cells using CRISPR-Cas9 and performed RNA-seq on coding RNA from DDX6 KO and WT cells. This data was used to determine if ISGs were upregulated in DDX6 KO HAP1 cells.
Project description:RNA-Seq reads and TopHat (Trapnell et al. Bioinformatics 2009) alignments of K562 cell-line transcriptome. These were used to validate the expression of short peptides idenitified by Mass-Spectrometry in K562 cells. K562 polyA+ RNA (Batch 1) and total RNA (batch 2) was purchased from Ambion. We used oligo (dT)-selected polyA+ RNA to construct libraries for RNA-Seq.We then profiled the transcriptome of polyadenylated mRNA-Seq using Illumina sequencing platforms. We then used the sequenced reads to reconstruct the transcriptome using the Cufflinks de-novo assembler (Trapnell et al. Nat.Bio.Tech. 2010). Recent computational and ribosome profiling analyses suggest that many short open reading frames (sORFs) in eukaryotic genomes are translated. However, evidence that these sORFs produce stable polypeptides is lacking. Here we develop a strategy to discover and validate novel sORF-encoded polypeptides (SEPs) in human cells. In total, we detect 117 SEPs, 114 of which are novel, varying in length from 15 to 149 amino acids. Of these, 10 SEPs (0.5%) are derived from long intergenic non-coding RNAs (lincRNAs). We also observe the presence of polycistronic genes and the widespread use of non-AUG start codons, which is a phenomenon historically thought to be rare in the mammalian genome. Quantitative measurements reveal that SEPs can be found at concentrations between ~10-2000 copies per cell, which is within the range of typical cellular proteins. We confirm the translation of a number of these SEPs through heterologous expression of their encoding cDNAs. We also discover that several SEPs possess properties characteristic of functional proteins. These results demonstrate that human sORFs produce numerous stable polypeptides, revealing that the human proteome is larger and more diverse than previously appreciated.
Project description:Expression microarray experiments were performed to identify all of the aerobic and hypoxic transcripts in wild-type cells. The role of Hap1 in the regulation of transcription was examined by monitoring gene expression in hap1 deletion cells. Keywords: gene expression, strain comparison, response to hypoxic conditions Overall design: Three replicates were performed for wild-type cells, and three replicates for hap1 deletion cells. Biological replicates were performed in each case, with separate cultures grown on different days. For each microarray, an aerobic sample was compared to a hypoxic sample.
Project description:The aim of this study is to analyze the change in genome wide expression levels in HAP1 cells upon loss of SMARCB1, SMARCA4 or both these genes together. The SMARCB1 and SMARCA4 genes were the hits from a genome wide screen involving genetrap mutagenesis to find new players that are involved in sensitivity to Doxorubicin (Dox). It was found that loss of SMARCB1 and SMARCA4 genes impart resistance in HAP1 cells to Dox. To validate this, the genes were knocked out in HAP1 cells with CRISPR-Cas9 technology. Gene expression levels in SMARCB1 null, SMARCA4 null and SMARCB1-SMARCA4 double null cells were compared to wildtype HAP1 cells using RNAseq. From these experiments it was found that SMARCB1 loss caused several fold increase in ABCB1 gene levels. ABCB1 is an efflux pump in cells responsible for flushing out many small-molecule drugs. Further analysis of this gene confirmed that ABCB1 was the main factor responsible for Dox resistance upon SMARCB1 loss. In total there are four different cell types with two replicates for each cell type. Therefore, 8 samples in total.
Project description:We used RNA-seq to monitor mRNA levels of all genes in response to hypoxia of wild-type yeast, S. cerevisiae (strain yMH914 with wildtype HAP1). To gain insights into how gene expression changes over time, cells were subjected to 100% nitrogen gas and collected after 0,5,10,30,60,120,180, and 240 minutes. Total RNA was extracted and mRNAs were enriched by polyA selection. The cDNA was prepared into a sequencing library, multiplexed and single-end sequenced by an Illumina HiSeq 2500 sequencer. After mapping with Tophat2, the number of reads per feature was calculated using HTSeq. RNA-seq analysis of eight time points of a yeast strain grown in hypoxia
Project description:The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long non-coding RNAs (lncRNAs) in the vasculature is largely unknown. Here, we characterized the expression of lncRNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1; also known as MALAT-1 or NEAT2). Endothelial cells of different origin express high levels of the conserved lncRNAs MALAT1, TUG1, MEG, linc00657 and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by siRNAs or GapmeRs induced a pro-migratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. If angiogenesis was further stimulated by VEGF, MALAT1 siRNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Gene expression profiling followed by confirmatory qRT-PCR demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro and its genetic deletion reduces neonatal vascular growth in vivo. Human endothelial polyA+ RNA expression analysis. We used two replicate samples of human umbilical vein endothelial cells.
Project description:Coilin iCLIP data revealed 42 novel human snoRNAs of intronic origin. To validate their expression and estimate abundance of novel and annotated snoRNAs, we performed RNA-seq on polyA- and rRNA-depleted RNA isolated from HeLa cells. Results show that expression of novel snoRNAs is comparable to the previously annotated snoRNAs. 1 replicate of RNA depleted of polyA and ribosomal RNA.