Digestion treatment of uscfDNA extracted by SPRI Method
Ontology highlight
ABSTRACT: We report the application of an uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma. The uscfDNA sequencing pipeline include two uscfDNA-optimized extraction methods (QiaM and SPRI) and one control extraction method (QiaC). Final libraries are made with a single-stranded library prepartion kit. We generate genome-wide maps revealing the presence of an uscfDNA population (25-99bp) in addition to the mncfDNA (100-250bp).
Project description:We report the application of an uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma. The uscfDNA sequencing pipeline include two uscfDNA-optimized extraction methods (QiaM and SPRI) and one control extraction method (QiaC). Final libraries are made with a single-stranded library prepartion kit. We generate genome-wide maps revealing the presence of an uscfDNA population (25-99bp) in addition to the mncfDNA (100-250bp).
Project description:We report the application of an uscfDNA-oriented sequencing pipeline profiling the cell-free DNA populations in plasma. The uscfDNA sequencing pipeline include two uscfDNA-optimized extraction methods (QiaM and SPRI) and one control extraction method (QiaC). Final libraries are made with a single-stranded library prepartion kit. We generate genome-wide maps revealing the presence of an uscfDNA population (25-99bp) in addition to the mncfDNA (100-250bp).
Project description:Here we have designed a full processing pipeline of P. falciparum RNA that innclues RNA preservation, extraction, cDNA synthesis and amplification, globin depletion and final transcriptome readout. To validate the protocol we have analyzed transcriptomes of P. falciaprum samples derived from filed studies as well as 3D7 lab strains.
Project description:Here we have designed a full processing pipeline of P. falciparum RNA that innclues RNA preservation, extraction, cDNA synthesis and amplification, globin depletion and final transcriptome readout. To validate the protocol we have analyzed transcriptomes of P. falciaprum samples derived from filed studies as well as 3D7 lab strains. This dataset have been used to establish the best conditions for whole transcriptome amplification protocol. Additionally it inclues the validation of bead extraction protocol. It also contains field samples used for validation of the entire protocol.
Project description:Human spermatozoa proteomics exposed to some physical, biological or chemical stressors is being explored. However, there is a lack of systematic comparative study on protein extraction methods to achieve in-depth coverage. Meanwhile, it is not clear if antibiotics can regulate proteins to affect sperm quality. Here we systematically compared and optimized the sperm protein extraction methods. It showed that the UA_Ultrasonication extraction method can make more kinds of protein to obtain higher protein extraction rate. Applying the optimized method, a largest human sperm proteome (5685 protein groups) from healthy human sperm samples was described. Besides, we continually investigated for the first time the differential sperm characteristics between medication (amoxicillin and clarithromycin) period and withdrawal period’s sperm samples obtained from a patient with Helicobacter pylori infection using semen examination, morphological analysis, bioinformatic analysis and data-independent acquisition tandem mass spectrometry (DIA-MS/MS). The longitudinal study result indicated that antibiotics can disrupt proteome expression in sperm, leading quantitative coverage of 159 differentially expressed proteins. The reliability of the results was verified by four proteins (ACTB, SCRN2, FDFT1, PLPP1) quantified by western blot. These proteins were mainly located at cytosol, involved in phosphorylation process, adenosine triphosphate (ATP) binding and metabolic pathways using bioinformatic analysis. This work provides an optimized extraction method to characterize the in-depth human sperm proteome and to extend its clinical applications. In addition, antibiotics should be avoided when it is necessary to preserve sperm quality for reproductive purpose.
Project description:MicroRNAs (miRNA) are non-coding RNAs that negatively regulate gene expression by preventing the translation of specific mRNA transcripts. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits with and without addition of a carrier. We were able to profile miRNA levels in serum samples using the small RNA sequencing method on the Illumina platform and observed that successful sequencing cannot be predicted by substrate RNA quality. Although the carrier RNA had a significant impact on miRNA measurement, it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios and higher numbers of processed reads, but the majority of the reads were not aligning to miRBase. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies and by careful selection of an extraction method, permitting archived serum samples to become valuable resources for high-throughput applications.
Project description:Genome-wide histone modification profiling using ChIP-seq of diabetic or non-diabetic murine bone marrow derived macrophages (BMDM) and haematopoietic stem cells (HSC). Chromatin fragments were selected using anti-Histone H3 (tri methyl K4) (ab8580) or anti-histone H3 (acetyl K27) (ab4729). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit. instructions. The final libraries were purified using SPRI Ampure XP beads to remove adaptor dimers and sequenced by the Biomedical Sequencing Facility (BSF) at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-read configuration.