Project description:Small RNAs isolated from RBCs were size fractionated by gel electrophoresis and used for the creation of 6 libraries. For the library from healthy children (library 1), RNA from 4 individuals was pooled. Libraries were multiplexed and analyzed on a 454 sequencing platform 26 yielding 569,621 sequence reads after demultiplexing into the 6 libraries
Project description:Small RNAs isolated from RBCs were size fractionated by gel electrophoresis and used for the creation of 6 libraries. For the library from healthy children (library 1), RNA from 4 individuals was pooled. Libraries were multiplexed and analyzed on a 454 sequencing platform 26 yielding 569,621 sequence reads after demultiplexing into the 6 libraries Analysis of 6 small RNA libraries from human red blood cells
Project description:Whole MeRIP-sequencing analysis was performed and analyzed by Lc. Aksomics Co. Ltd. (Shanghai, China). The peri-infarct cortex from sham mice, PT mice with EV-Vector, and PT mice with EV-circSCMH1 was collected in TRIzol. Total RNAs were qualified by agarose gel electrophoresis and quantified using Nanodrop. mRNA was isolated and then fragmented to 100-nucleotide-long fragments. m6A methylated mRNAs were enriched with anti-N6-methyadenosine(m6A) antibody. We use commercial kit for RNA-seq library prepa ration of m6A mRNA and input samples. Completed libraries were qualified and then sequenced on Hiseq 4000 platform.
Project description:Urine from a patient with a urinary tract infection was plated on LB agar plate. Microcolonies appeared ~8h after plating. Microcolonies were picked and subjected to Microcolony-seq and to whole-genome sequencing. Genomic DNA of each UTI microcolony was extracted from 1 mL of bacteria using the DNeasy Blood & Tissue Kit (Qiagen). Library preparation and sequencing was carried out by BGI company, China. Concentration of samples was detected by fluorometer or Microplate Reader (Qubit Fluorometer, Invitrogen). Sample integrity and purity were detected by Agarose Gel Electrophoresis. 1μg genomic DNA was randomly fragmented by Covaris. The fragmented genomic DNA were selected by Agencourt AMPure XP-Medium kit to an average size of 200-400bp. Fragments were end repaired and then 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated fragments. PCR products were purified by the Agencourt AMPure XP-Medium kit. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. Samples were deep-sequenced with the DNBseq G400 machine using the 150-cycles paired-end with 350 bp insert size. At least 150X sequencing depth for each nucleotide in each sample was targeted.
Project description:Next-generation RNA sequencing (RNA-seq) is hampered by “primer dimer” (PD) artifacts and its quantitative performance reduced by polymerase fall-off (PF) at RNA modifications and secondary structures. Here we improved RNA-seq efficiency by incorporating (i) a post-reverse-transcription (RT) digestion of excess primers with Escherichia coli exonuclease I for PD mitigation, thus obviating gel purification during RNA-seq library preparation, and (ii) a high-processivity reverse transcriptase to increase full-length reads. A full factorial experimental design was applied to absolute quantification RNA sequencing (AQRNA-seq), the most accurate NGS-based method for quantifying small RNAs, using cDNA libraries constructed from E. coli small RNAs (>85% tRNA) followed by sequencing, data processing, and data analysis. The novel PF and PD mitigation approaches increased AQRNA-seq sensitivity >10-fold by minimizing PF and maximizing target RNA reads. By increasing sensitivity and obviating gel electrophoresis for removing PD, AQRNA-seq and other NGS-based RNA-seq methods can now be automated to increase throughput and reduce RNA sample size.
Project description:In this research, capillary electrophoresis was combined with liquid chromatography to sequence novel monoclonal antibodies. It was demonstrated CE can provide highly complemetary information to LC in the process of novel mAbs sequencing.
Project description:This dataset submission includes the raw files used to sequence a mix of 5 human monoclonal antibodies as a benchmark proof-of-concept study for the manuscript "de novo Protein Sequencing of Antibodies for Identification of Neutralizing Antibodies in Human Plasma Post SARS-CoV-2 Vaccination". Refer to the attached metafile for detailed descriptions of the experiments. Briefly, bottom-up data was generated by performing in-solution digestions on the monoclonal antibodies on their own and after mixing to generate a pseudo-polyclonal mix. Separation was performed using native gel and non-reducing SDS-PAGE (NRT), followed by in-gel digestion for heavy-light chain pairing. Lastly, C-terminal modification and EThcD fragmentation of in-solution digests allowed for I/L determination.
Project description:Currently, the genomes of many toxin proteins remain unsequenced. Characterizing these toxin proteins and their active peptide segments is crucial for the development of detoxification agents and for discovering proteins with potential therapeutic applications. Existing methods for identifying and detecting toxin proteins primarily focus on known proteins, making them less applicable for identifying toxins from unknown sources. In this study, we employed a combination of multiple enzymatic digestion techniques, de novo mass spectrometry sequencing, and sequence assembly methods based on graph theory to perform high-precision, full-length sequencing of multi-subunit toxin proteins. After de novo sequencing and identifying Ricin toxin proteins, we utilized a strategy based on multiple alignments and error correction through the construction of overlapping scaffolds to differentiate between homologous isomers I and L and to detect unexpected post-translational modifications. This approach, referred to as the Heuristic Denovo protein sequencing(HDPS), facilitated the rapid hydrolysis and identification of proteins. The method achieved a 100% coverage rate for protein/peptide biological toxins and an amino acid-level accuracy of over 95%. Through de novo sequencing of Ricin toxin, this study provides a stable and reliable solution for identifying highly mutated proteins and those from unknown sequence sources.
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track is produced as part of the ENCODE project. The track reports the percentage of DNA molecules that exhibit cytosine methylation at specific CpG dinucleotides. In general, DNA methylation within a gene's promoter is associated with gene silencing, and DNA methylation within the exons and introns of a gene is associated with gene expression. Proper regulation of DNA methylation is essential during development and aberrant DNA methylation is a hallmark of cancer. DNA methylation status is assayed at more than 500,000 CpG dinucleotides in the genome using Reduced Representation Bisulfite Sequencing (RRBS). Genomic DNA is digested with the methyl-insensitive restriction enzyme MspI, small genomic DNA fragments are purified by gel electrophoresis, and then used to construct an Illumina sequencing library. The library fragments are treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymidine while leaving methylated cytosines intact. The sequenced fragments are aligned to a customized reference genome sequence and for each assayed CpG we report the number of sequencing reads covering that CpG and the percentage of those reads that are methylated. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf DNA methylation at CpG sites was assayed with a modified version of Reduced Representation Bisulfite Sequencing (RRBS; Meissner et al., 2008). RRBS was performed on cell lines grown by many ENCODE production groups. The production group that grew the cells and isolated genomic DNA is indicated in the "obtainedBy" field of the metadata. When a cell type was provided by more than one lab, the data for the cells from only one lab are displayed in the table above. However, the data for every cell type from every lab is available from the Downloads page. RRBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of genomic DNA Genomic DNA is isolated from biological replicates of each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation are determined using fluorescent DNA binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 1 µg of DNA is used to make an RRBS library; however, we have also had success in making libraries with 200 ng genomic DNA from rare or precious samples. RRBS library construction and sequencing RRBS library construction starts with MspI digestion of genomic DNA , which cuts at every CCGG regardless of methylation status. Klenow exo- DNA Polymerase is then used to fill in the recessed end of the genomic DNA and add an adenosine as a 3prime overhang. Next, a methylated version of the Illumina paired-end adapters is ligated onto the DNA. Adapter ligated genomic DNA fragments between 105 and 185 basepairs are selected using agarose gel electrophoresis and Qiagen Qiaquick Gel Extraction Kit. The selected adapter-ligated fragments are treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite treated DNA is amplified in a final PCR reaction which has been optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction uracils are copied as thymines resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. The sample is now ready for sequencing on the Illumina sequencing platform. These libraries were sequenced with an Illumina Genome Analyzer IIx according to the manufacturer's recommendations. Data analysis To analyze the sequence data, a reference genome is created that contains only the 36 base pairs adjacent to every MspI site and every C in those sequences is changed to T. A converted sequence read file is then created by changing each C in the original sequence reads to a T. The converted sequence reads are aligned to the converted reference genome, and only reads that map uniquely to the reference genome are kept. Once reads are aligned the percent methylation is calculated for each CpG using the original sequence reads. The percent methylation and number of reads is reported for each CpG.
Project description:We are submitting raw data files used for de novo sequencing of human polyclonal antibodies targeting receptor binding domain (RBD) from covid-19 virus to generate recombinantly produced monoclonal antibodies capable of neutralizing RBD. The raw files submitted are described in the metafile included, but in brief, they are categorized as data generation (i.e., bottom-up in-solution enzymatic digestion), native gel separation followed by digestion, non-reducing room temperature (NRT) gel separation followed by digestion, middle-down, non-reducing, and isobaric resolution.