Takara SMART-Seq® Human TCR (with UMIs)

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 µg 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.

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+ T‑cell 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.

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.

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 TCRα and TCRβ 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 (1–96) 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 Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (97–192) 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 Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (193–288) 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 Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (289–384) 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 Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (1–24) contains 24 pairs of unique dual-indexed PCR primers, which are a subset of the Unique Dual Index Kit (1–96), Cat. No. 634752. They can be used to generate up to 24 Illumina-compatible sequencing libraries and are recommended with several of Takara Bio’s RNA-seq and DNA-seq kits.