Project description:A cDNA library was constructed by Novogene (CA, USA) using a Small RNA Sample Pre Kit, and Illumina sequencing was conducted according to company workflow, using 20 million reads. Raw data were filtered for quality as determined by reads with a quality score > 5, reads containing N < 10%, no 5' primer contaminants, and reads with a 3' primer and insert tag. The 3' primer sequence was trimmed and reads with a poly A/T/G/C were removed
Project description:Raw RNA sequencing data derived from human CAR-T cell samples are deposited in this repository. The datasets include bulk single-cell RNA-seq reads generated for research purposes.
Project description:Label-free protein sequencing is critically enabled by bottom-up, mass spectrometry-based proteomics workflows. Applications such as antibody sequencing or antigen discovery require de novo reconstruction of peptide and protein sequences. While trypsin has long served as the gold-standard protease in proteomics, its restricted C-terminal cleavage specificity constrains peptide diversity, particularly limiting coverage in antibody hypervariable complementarity-determining regions (CDRs). As a result, current workflows yield sparse reads and sequence gaps. Although multi-protease and hybrid-fragmentation strategies can notably improve coverage, they add complexity and compromise scalability and reproducibility. Here, we present a novel approach using HyperThermoacidic Archaeal (HTA) proteases Krakatoa or Vesuvius as powerful single-enzyme solutions for de novo antibody sequencing. Each protease generated over five times more unique peptide reads than trypsin or chymotrypsin with high redundancy across CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this workflow enabled complete, unambiguous antibody sequencing. Despite most de novo tools being optimized for CID/HCD-tryptic data, analysis using PEAKS/DeepNovo and Stitch softwares showed that HTA-Proteases yielded up to fourfold higher alignment scores and fewer sequence mistakes across variable regions. Redundant reads increased more than threefold compared to standard proteases, boosting confidence in amino acid assignment and reducing ambiguity in final assemblies. Our alternative HTA-EAciD approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This single-protease strategy delivers sequencing performance comparable to multi-enzyme workflows, providing a scalable, efficient, and highly confident approach for de novo sequencing in antibody discovery and beyond.
Project description:We use nucleosome maps obtained by high-throughput sequencing to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a secondary role. Furthermore, similar sequence signatures are exhibited by control experiments in which genomic DNA is either sonicated or digested with micrococcal nuclease in the absence of nucleosomes, making it possible that current predictions based on highthroughput nucleosome positioning maps are biased by experimental artifacts. Included are raw (eland) and mapped (wig) reads. The mapped reads are provided in eland and wiggle formats, and the raw reads are included in the eland file. This series includes only Mnase control data. The sonicated control is part of this already published accession, as is a in vitro nucleosome map: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15188 We also studied data (in vitro and in vivo maps as well as a model) from http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13622 and from: http://www.ncbi.nlm.nih.gov/sra/?term=SRA001023