12/2/2023 0 Comments 10x chromium system![]() Supplementary files format and content: Transcriptomic and HashTag (HTO) counts matrices in Matrix Market sparse CSC matrix format. This produces a matrix of counts with hashtag labels for rows and cell barcodes for columns. ![]() If present, I1 contains the index which is needed for 10X CellRanger pipelines.Īn unfiltered digital gene expression matrix was generated for each gene expression library against the 10X Genomics mouse reference genome (mm10-2020-A) build using CellRanger v4.0.0Ī digital counts matrix was generated for each hashtag library using CITE-Seq-Count. The raw data (FASTQ file) of each library were produced and demultiplexed using Illumina bcl2fastq 2.20.0.422.įor all libraries, R1 contains a 16bp cell barcode + 12bp UMI barcode and R2 contains the biological reads. cDNA and libraries were checked for quality on an Agilent 4200 TapeStation, quantified by KAPA qPCR. cDNA was synthesized and amplified as per manufacturer’s protocol and amplified to construct an Illumina sequencing library. Single cell capture, barcoding and library preparation were performed with the 10x Chromium system using version 3 chemistry, following the manufacturer’s protocol. Cells capture, lysis and RNA extraction were performed using the 10X V3 Chromium technology following the manufacturer’s protocol. Around 24,000 cells (8,000 cells from each hashtagged sample) were loaded into one lane of a 10X Chromium microfluidic chip. Neutrophils were sorted from BM, PB and lung, and then labeled with cell hashing antibodies. Neutrophils from naïve (C57BL/6J) mice, gene expression library This service can be performed in conjunction with the 5′ Next GEM library prep or the VDJ immune profiling (B cell or T cell) library prep, but is not a stand alone service.GEO help: Mouse over screen elements for information. It can also be used to multiplex more than one sample into a single library prep. This technology can also determine antigen specificity of single T cells with Feature Barcode peptide-MHC multimers to study the dynamic interactions between lymphocytes and antigens. Use this technique to measure both gene and cell surface protein expression in the same cell to identify protein isoforms, detect protein for low abundance transcripts, and further increase phenotypic specificity. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions. It does so by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. A pool of ~750,000 barcodes are sampled separately to index each cell’s transcriptome and cell surface protein. This is accomplished by labeling cell surface proteins with antibodies conjugated to a Feature Barcode oligonucleotide, followed by direct capture of the Feature Barcode by the Gel Bead primer. ![]() The Chromium Single Cell Feature Barcode technology offers a comprehensive, scalable approach to detect cell surface proteins along with the gene expression and immune repertoire information from the same single cell. UVA Child Development & Rehabilitation Center.Plastic Surgery, Maxillofacial, & Oral Health.Translational Health Research Institute of Virginia.Institute of Law, Psychiatry & Public Policy.Child Health Research Center (Pediatrics).Thaler Center for AIDS & Human Retrovirus Research Center for Immunity, Inflammation & Regenerative Medicine.Center for Behavioral Health & Technology.Molecular Physiology & Biological Physics. ![]() Microbiology, Immunology, & Cancer Biology (MIC). ![]()
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