Project description:This experiment was designed to improve the proportion of miRNA mapping reads from sRNA library sequencing, by reducing the amount of particularly abundant rRNA fragments.
Project description:This experiment was designed to improve the percentage of miRNA mapping reads from sRNA library sequencing, by reducing the amount of one particularly abundant rRNA 30mer sequence.
Project description:We report the different expression level of in vivo transcriptome and DMEM cultured bacteria E.coli O157:H7 samples. After obtaining RNA from mice colon and LB cultured free living cells, RNA samples were purified using an RNeasy Mini Kit (Qiagen) and bacterial rRNAs were depleted using the Ribo-Zero rRNA Removal Kit (Epicentre; #RZNB1056). Library construction of RNA samples was carried out using a NEBNext R UltraTM Directional RNA Library Prep Kit for Illumina R (NEB, USA), following the manufacturer’s recommendations. After cluster generation, library preparations were sequenced on an Illumina Hiseq platform to generate paired-end reads.We find that a novel sRNA s77 significantly upregulated compared in vivo samples and LB-cultured sample. This study provides a novel sRNA for further identifications.
Project description:Small RNAs (sRNAs) play important roles in plants encountering stress environments. However, limited research has been conducted on the sRNAs involved in plant wound responses. To identify potential roles for the wounding-related sRNAs, sRNA deep sequencing was used. After leaves were wounded for 0.5 hour, total RNAs from unwounded and wounded leaves were isolated for sRNA library construction. The Illumina platform was used to sequence sRNA libraries. About 12 million sequence reads were obtained for each sample.
Project description:Genome-wide mapping and characterization of novel Notch-regulated long non-coding RNAs in acute leukemia Total RNA was extracted from samples using the RNeasy Plus mini kit (Life Technologies, Carlsbad, CA). Samples were then subject to PolyA selection (Figures 1E, 5F and 5G only) using oligo-dT beads (Life Technologies, Carlsbad, CA) or rRNA removal (all other samples) using the Ribo-Zero kit (Epicentre, Madison, WI) according to the manufacturers instructions. The resulting RNA samples were then used as input for library construction using the dUTP method as described by Parkhomchuck et al, 2009. RNA libraries were then sequenced on the Illumina HiSeq 2000 or 2500 using 50bp paired-end reads.
Project description:A spectral library was built for Drosophila melanogaster. The spectral library allows reproducible quantification for thousands of peptides per SWATH-MS analysis.
Proteins from Drosophila melanogaster embryo, adult flies were digested with trypsin using in-gel digestion and the peptides were fractionated by high-pH reverse phase chromatography. HRM peptides were spiked into the peptides mixture and each fraction was injected on a Sciex TripleTOF 6600 mass spectrometer fitted with microflow set-up.
The resulting .wiff files were analysed using MaxQuant and Spectronaut.
Project description:High-throughput sequencing (HTS) has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA) and other small RNAs (sRNA). Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps; specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material. 28 samples were sequenced and the libraries were made using various synthetic oligo mixtures and adaptor combinations