Project description:ChIP-Seq for H3K27 trimethylation was performed for two HPV-positive and two HPV-negative squamous cell carcinoma cell lines. The data served two purposes. First, the data were used as an example implementation of our novel ChIP-Seq Peak Prioritization pipeline, PePr. We have developed the PePr pipeline, a ChIP-Seq Peak Prioritization pipeline that accounts for the variation among replicates and peak location relative to a gene. We show, using a transcription factor dataset (which exhibited small variation among samples), that PePr performs favorably compared to commonly used peak callers and that it achieves balanced sensitivity and specificity. We also show, using histone modification data (which exhibited larger variation among samples), that PePr can improve the detection of differential H3K27me3 regions compared with a common current approach. Using data from ChIP-Seq and gene expression experiments performed in parallel on the same samples, we show that the incorporation of functional annotations can improve the prioritization of functional sites. Secondly, the data were used to assess real differences in the genome-wide H3K27me3 profiles between HPV-positive and HPV-negative carcinoma cell lines. Careful analysis and integration of the data with DNA methylation and gene expression data performed on the same cell lines demonstrated striking differences exist. ChIP-Seq for H3K27 trimethylation was performed for two HPV-positive and two HPV-negative squamous cell carcinoma (SCC) cell lines. Input DNA was also sequenced for each sample to serve as a control. The goal was to determine overall differences in H3K27me3 patterns observed between the HPV-positive and HPV-negative SCC cell lines.
Project description:UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae ) and bacteria (E. coli). In all analyses, SILAC labeling was used to quantify protein recovery in the presence and absence of irradiation. For S.cerevisiae, we also compared crosslinking using 254nm (UVC) irradiation (TRAPP) with 4-thiouracil (4SU) labeling combined with 350nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was significantly higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its strong, but largely sequence independent, binding to RNA in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems. Overall design: CRAC datasets were collected for Eno1 and an untagged control (BY). Two replicates were collected for each.
Project description:Raw files corresponding to five immunocyte populations deep sequenced using ImmGen's standard ultra-low input RNA-seq pipeline Overall design: Samples collected by one ImmGen lab and sequenced using ImmGen's standard RNA-seq pipeline contributor: Immunological Genome Project Consortium
Project description:Raw files corresponding to diverse mononuclear phagocytes populations sequenced using ImmGen's standard ultra-low input RNA-seq pipeline Overall design: Samples collected by 16 labs from 9 sites, shipped frozen to a central location, and sequenced using ImmGen's standard RNA-seq pipeline Contributor: Immunological Genome Project Consortium