Project description:Urine was obtained from a patient with a urinary tract infection and frozen in 15% glycerol at -80C. For the Microcolony-seq experiment a sample from the frozen urine was serially diluted four times 10-fold and plated on an LB agar plates and incubated at 37C. After 8 hours of incubated microcolonies were visible. Twenty microcolonies were picked and subjected to the Microcolony-seq pipeline and four additional microcolonies were mixed and separated to four Eppendorf tubes and served as the technical replicates of the experiment. RNA was extracted with 1 mL TriReagent (Sigma-Aldrich) per sample. RNA quality was assessed by Nanodrop and by Bioanalyzer using the Agilent RNA 6000 Pico Kit (5067-1513). rRNA depletion was done by using the DIY rRNA depletion method, using the rRNA sequence probe set designed for E. coli K-12. RNA-seq libraries were constructed based on the RNAtag-Seq protocol with several modifications. The rRNA depletion step was done first following by the fragmentation step in the RNAtag-Seq protocol. Then the first ligation was carried out and the rest of the RNAtag-Seq protocol was followed. Libraries were single-end sequenced using the Nextseq500 Sequencer (Illumina).
Project description:We developed a cost-effective enzyme-based rRNA-depletion method tailored for Drosophila melanogaster, addressing the limitations of existing commercial kits and the lack of peer-reviewed alternatives. Our method employs single-stranded DNA probes complementary to Drosophila rRNA, forming DNA-RNA hybrids. These hybrids are then degraded using RNAse H enzyme, effectively removing rRNA and enriching all non-ribosomal RNAs, including mRNA, lncRNA and small RNA. When compared to a commercial rRNA Fly Kit, our approach demonstrated superior rRNA removal efficiency and mapping percentage, confirming its effectiveness. Additionally, our method successfully enriched the non-coding transcriptome, making it a valuable tool for studying ncRNA in Drosophila. The probe sequences and rRNA-depletion protocol is made freely available, offering researchers a reliable alternative for rRNA-depletion experiments.
Project description:We performed RNA-seq experiments on a total of 12 mouse immune organs, including spleen (SP), bone marrow (BM), lymph node (LN) and Peripheral blood mononuclear cell (PBMC). Briefly, RNA-Seq libraries were constructed after rRNA depletion using a NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB). The E6310L NEBNext Ultra RNA Library Prep Kit for Illumina(NEB, E7530S)(NEB) was used according to the manufacturer’s instructions and the cDNAs were sequenced with the Hiseq X10 platform(Illumina)
Project description:These experiments were designed to quantify depletion of rRNA sequencing reads from bacterial RNA-seq libraries and verify that mRNA sequencing reads were not altered. Specifically, we tested an rRNA depletion method using custom-designed biotinylated oligonucleotides and compared these results to undepleted (total RNA) libraries and libraries made with the previously-available Ribo-Zero kit (Illumina).