SMART-Seq Human TCR (with UMIs)

Description

SMART-Seq Human TCR with UMIs enables users to analyze T-cell receptor TCR repertoires from bulk RNA samples. This kit can generate libraries from total RNA 10 ng to 1 mug from peripheral blood leukocytes, 20 ng to 200 ng of total RNA obtained from whole blood, or 1 ng to 100 ng T cells or purified T cells 1,000 to 10,000 cells and can be used to generate data for both TCR-alpha or TCR-beta subunits. Unique molecular identifiers UMIs are incorporated to facilitate PCR error correction and clonotype quantification during data analysis, and unique dual indexes UDIs allow users to multiplex more samples and provide confidence when sequencing on patterned flow cells. The kit generates indexed libraries ready for sequencing on Illumina platforms. Easily analyze and visualize your data with Cogent NGS Immune Profiler Software and Cogent NGS Immune Viewer tool. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back Back Variable and highly complex samples are accommodated without the need for RNA purification or T-cell enrichment.TRA and TRB libraries were generated from 1, 10, and 100 ng of human CD3+ Tcell total RNA Panel A , 1,000 and 10,000 CD3+ T cells Panel B , and 20 ng of whole-blood RNA extracted from three different samples Panel C . The sequence reads were processed by Cogent NGS Immune Profiler. Back The number of clonotypes detected reveals biological variation.Duplicate libraries were generated from 10 ng RNA extracted from single-donor PBMC samples P1-P6 and sequenced on an Illumina MiSeq system using 300-bp paired-end reads to obtain approximately 1.5 million reads per sample. Resulting sequencing reads were processed with Cogent NGS Immune Profiler. Clonotype numbers from different donors for TRA blue and TRB orange are shown. Error bars shown represent the standard error between the duplicates. Back Back Schematic of technology and workflow for SMART-Seq Human TCR with UMIs .Input RNA or cells are oligo-dT primed using the TCR dT Primer dark blue . Following oligo dT-priming, SmartScribe Reverse Transcriptase performs first strand cDNA synthesis on input RNA or cells and adds non-templated nucleotides to the 5 end of each cDNA molecule XXXXX . Upon reaching the 5 end of the RNA template, the TCR SMART UMI Oligo anneals to the non-templated nucleotides XXXXX , incorporating UMI yellow and partial Illumina adapter light green complementary to the hTCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify TCR cDNAs. In PCR1, the hTCR PCR1 Universal Forward primer anneals to the complementary sequence carried by the TCR SMART UMI Oligo light green , incorporating the Illumina Read2 sequence dark green . hTCRa PCR1 reverse and hTCRb PCR1 reverse primers orange anneal to sequences in the constant regions of TCRalpha and TCRbeta cDNA, respectively, to amplify the entire TCR V D J region. During PCR2, the nested hTCRa and/or hTCRb PCR2 UDI reverse primers anneal to sequences in TCR constant regions that are internal to the sequences bound by the hTCRa/b PCR1 reverse primers and adds the Illumina Read 1 sequence light purple . In the same PCR2 reaction, Unique Dual Index Kit primers anneal to the sequence added by hTCR PCR1 Universal Forward primer or the TCRa and/or TCRb PCR2 UDI reverse primers to add Illumina P7-i7 and P5-i5 index sequences dark blue . The result is a sequencing-ready library that contains the entire TCR variable region with a small portion of the constant region. Required Products The Unique Dual Index UDI Kit 196 contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bios RNA-seq and DNA-seq kits. Back The Unique Dual Index UDI Kit 97192 contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bios RNA-seq and DNA-seq kits. Back The Unique Dual Index UDI Kit 193288 contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bios RNA-seq and DNA-seq kits. Back The Unique Dual Index UDI Kit 289384 contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bios RNA-seq and DNA-seq kits. Back The Unique Dual Index UDI Kit 124 contains 24 pairs of unique dual-indexed PCR primers, which are a subset of the Unique Dual Index Kit 196 , Cat. No. 634752. They can be used to generate up to 24 Illumina-compatible sequencing libraries and are recommended with several of Takara Bios RNA-seq and DNA-seq kits. Back