3' isoform specific RNA IP (3' isRIP-seq) for PUF3 RBP in budding yeast
ABSTRACT: Exponentially growing cells (OD 0.5-0.8) from the TAP-tagged strain were harvested by ultracentrifugation followed by of washing and flash freezing in liquid nitrogen and breakage in FastPrep24 (ZYmoresearch S6005). The lysate was subjected to IP with Dyna M280 sheep anti rabbit IgG beads for 2h at 4C. An aliquot was taken before the IP as an input control. The IP of the RNA-protein complex was subjected to cleavage by TEV protease to cleave the TAP-tag off the protein. The bound RNA was then precipitated in phenol/chloroform/isoamyl-alcohol and sequenced using 3' T-filling protocol as described by Wilkening et al 2013 to obtain accurate quantification of 3' isoforms. We used the R Bioconductor package DESeq2 (Anders and Huber 2010) to call for differential log change between the Input and the RNA-IP sample and took all those isoforms as differentially regulated which had more than 4-fold change at a FDR of 10% as reported by the package.
Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3' transcript isoforms in yeast. Our results reveal widespread differences in stability ...[more]
Project description:The following data contains sequencing files for RNA IP and Polysome profiling experiments in wt and not5delta strains with background of Not1-TAP tagged strain (MAT_ leu2_20 ura3_ met15_ lys2_ his3delta1 not1::NOT1-Tap-tag-URA3). The RNA IP experiments have been performed as described previously (Gupta et al 2014) and the Polysome profiling experiments have been performed as described in (Albanese 2010). The libraries have been sequenced using polyadenylation isoform specific profiling protocol. The Not1 RIP experiments demonstrate genome-wide association of RNA with the central essential scaffold of the Ccr4-Not complex. The same RIP experiment in the not5delta background demonstrates the specificity of not5 in determining Not1 binding specificity to pulled down RNA. The polysome profiling experiments demonstrate the role of not5 in determining the translatability of ribosomal RNA.
Project description:Transcriptional arrest is performed in the rpb1-1 strain grown at 24C in two independent biological replicates by raising the temperature instantly to 37C. Total RNA was collected at fixed time points after the arrest and sequenced using the 3' T-filling protocol as described by Wilkening et al 2013 to obtain accurate quantification of 3' isoforms. The decaying library size is corrected for the by reads mapping to the S.pombe spike-in control RNA. The decay rates are estimated on fitting a single parameter exponential decay curve after taking the technical reproducibility of low counts for every time point into account.
Project description:HEK T293 cells were treated with Anisomycin or the solvent control DMSO. Cells were fractionated and cytoplasmic and nuclear RNA were isolated. pre-mRNA-Seq was performed on stressed and unstressed nuclear RNA samples. mRNA-Seq was performed on stressed and unstressed cytoplasmic RNA samples. In addition, polyadenylation sites were globally mapped by applying the 3'T-fill method on the same stressed and unstressed cytoplasmic RNA samples.
Project description:Whi3 associated mRNAs were identified by immunoprecipitation of TAP-tagged Whi3 followed by microarray analysis RNA IP of Whi3-TAP and Whi3-dRRM-TAP with total RNA as reference. Each sample has 2 replicates
Project description:Yeast RNA polymerase (Pol) II consists of a ten-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immuno-precipitation coupled to high-resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle. Keywords: ChIP-chip Comparison of Rpb3 vs. Rpb4/7 genome-wide occupancy profiles. Data obtained from independent ChIP-chip experiments on two yeast strains: S288C Rpb3TAP vs. Rpb7-TAP strain and W303 Rpb3-TAP vs. wild type strain (IP with the monoclonal antibody for Rpb4/7). Biological replicates W303 strains: 1 x Rpb3-TAP, 1 x wt with Rpb4/7 antibody, independently grown and harvested. Biological replicates S288C strains: 3 x Rpb3-TAP, 2 x Rpb7-TAP, independently grown and harvested. One replicate per array.