Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Picea abies tissues (including needles, immature cones and lateral bud meristem). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from needles, immature female cones and lateral bud meristem of Picea abies. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank David Neale for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:The goal of this experiment is to assess tissue preferential transcript accumulation and fold difference between two tissues that support secondary vascular growth in three spruces: Picea glauca, Picea sitchensis and Picea mariana. Tissues compared are secondary xylem (wood forming tissue located on the internal side of the cambial meristem) and phelloderm (composite sample of the phloem and phelloderm tissues located on the outer side of the cambial meristem). One-color comparison of 3 spruce species in 2 tissue types: xylem and phelloderm. 20 biological repetitions per tissue for Picea glauca and 15 for Picea sitchensis and Picea mariana, for a total of 100 slides.
Project description:We report the sequences bound to CENP-A in the dog genome (Canis familiaris) for high-throughput characterization of centromeric sequences. We compare these ChIPSeq reads (72 bp, single read) against a reference centromeric satellite DNA domain database for the dog genome, resulting in the annotation of sequence variation and estimated abundance of seven satellite families together with adjacent, non-satellite sequences. To study global patterns of sequence diversity and characterizing the subset of sequences correlated with centromere function, these sequences were evaluated relative to a comprehensive centromere sequence domain k-mer library. From this analysis, we identify functional sequence features from two satellite families (CarSat1 and CarSat2) that are defined by distinct arrays subtypes. Sequences bound to CENP-A in MDCK (dog) cell line
Project description:Analysis of the subunits composition of the thylakoids protein complexes in Picea abies (Norway spruce) by means of two-dimensional large-pore Blue-Native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D lpBN/SDS-PAGE) and in-gel tryptic digestion of single spots.
Project description:In this study, we sequenced small RNA content from seven major tissues/organs employing Illumina technology. More than 154 million reads were generated using Illumina high-throughput sequencing GAII platform, which represented more than 20 million distinct small RNA sequences. After pre-processing, several conserved and novel miRNAs were identified in chickpea. Further, the putative targets of chickpea miRNAs were identified and their functional categorization was analyzed. In addition, we identified miRNAs exhibitng differential and specific expression in various tissues/organs. We collected different tissue samples used in this study and total RNA isolated was subjected to Illumina sequencing. The sequenced data was further filtered using NGS QC Toolkit to obtain high-quality reads. The filtered reads were pre-processed using modified perl script provided in the miRTools software. After quality control, the identical reads were collapsed into a unique read and read count for each sequence was recorded. All the filtered unique reads from each sample were screened stepwise against annotated non-coding RNA sequences, including plant snoRNA, tRNA and rRNA. The remaining reads were screened against repeat sequences from RepBase and chickpea chloroplast sequence. Conserved miRNAs were identified based on similarity with miRBase database and novel miRNAs were identified using miRDeep-P pipeline. For differential expression analysis, the read count for each miRNA was normalized using DESeq software. The genes preferentially and specifically expressed in various tissues/organs were identified.