Takara SMART-Seq® Mouse TCR (with UMIs)

SMART-Seq Mouse TCR (with UMIs) enables users to analyze mouse T-cell receptor (TCR) diversity using purified high-integrity RNA (RIN>7) or whole intact cells as input. This kit contains enough reagent for 96 reactions.

Schematic of technology and workflow for SMART-Seq Mouse 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 mTCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify TCR cDNAs. In PCR1, the mTCR 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). mTCRa PCR1 reverse and mTCRb 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 the PCR2, the nested mTCRa and/or mTCRb 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 sequence added by the 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.

Evaluation of SMART-Seq Mouse TCR (with UMIs) performance across various sample types.Panel A.TCR libraries were made for both alpha (blue) and beta (purple) chains in 10, 100, 250, 500, and 1,000 ng of mouse spleen RNA with the SMART-Seq Mouse TCR (with UMIs) (mTCRv2) workflow. The resulting TCR libraries were sequenced on the Illumina NextSeq platform, generating 2 x 150 bp reads. Sequencing outputs were down sampled to ~1.5 x 107reads.Panel B.The mTCRv2 workflow was performed using 200 ng RNA isolated from four different sample types. The resulting TCR libraries were sequenced on NextSeq, generating 2 x 150 bp reads. Sequencing outputs were down sampled to 2.0 x 107reads for each sample type. Data was processed using Cogent NGS Immune Profiler software, and bar plot was generated to show clonotype counts for each individual input amount or sample type. TRA = TCRα chain, TRB = TCRβ chain.

Correction of PCR duplicates and sequencing errors using UMIs. Panel A.TRA and TRB sequencing libraries from 10 ng of mouse spleen RNA were prepared using the SMART-Seq Mouse TCR (with UMIs) (mTCRv2) workflow. TRA and TRB clonotype counts at different sequencing depths are shown with (blue line) and without (purple line) UMI-based error correction.Panel B.Venn diagrams show ~87% clonotype overlap between TRA libraries sequenced with different cartridges and ~88% clonotype overlap between TRB libraries sequenced with different cartridges.

Evaluation of sequencing flexibility withSMART-Seq Mouse TCR (with UMIs).To evaluate whether SMART-Seq Mouse TCR (with UMIs) (mTCRv2) can support sequencing full-length V(D)J or CDR3 only sequencing, TCR sequencing libraries generated with 10 ng or 100 ng of mouse spleen RNA were sequenced on the Illumina MiSeq® platform. Sequencing was done with either 2 x 150 bp reads (300 cycles) for CDR3 region or 2 x 300 bp reads (600 cycles) for V(D)J region. Sequencing outputs were down sampled to 1 x 106reads. Data was processed using Cogent NGS Immune Profiler Software. bar plot was generated to compare TRA and TRB clonotype counts for each input amount when sequenced with 2 x 150 bp or 2 x 300 bp reads.