Takara SMART-Seq Human BCR

SMART-Seq Human BCR (with UMIs) generates oligo(dT)-primed, full-length mRNA-seq libraries for all BCR heavy (IgG/m/D/E/A) and light (IgK/L) chains. The incorporation of unique molecular identifiers (UMIs) provides greater accuracy for quantitative gene expression analysis across samples while controlling for PCR errors and amplification biases. Up to 384 multiplexed, Illumina-ready sequencing libraries can be obtained using theUnique Dual Index Kits(Cat. # 634752–56). This kit offers an end-to-end solution including cDNA synthesis, library preparation, and data analysis with our free Cogent NGS bioinformatics tools.

Sensitive and reproducible clonotype detection from a broad range of RNA amounts. Human BCR heavy chain (IgA/D/E/g/m) and light chain (IgK/L) libraries were generated from 10, 100, and 1,000 ng of total human PBMC RNA using SMART-Seq Human BCR (with UMIs). The sequence reads were processed using Cogent NGS Immune Profiler Software.

Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs).Input RNA or cells are oligo-dT primed using the 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 SMART UMI Oligo anneals to the non-templated nucleotides (XXXXX), incorporating the UMI (yellow) and partial Illumina adapter (light green) complementary to the hBCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify BCR cDNAs. In PCR1, the hBCR PCR1 Universal Forward primer anneals to the complementary sequence carried by the SMART UMI Oligo (light green), incorporating the Illumina Read2 sequence (dark green). The hBCR PCR1 Reverse primer (orange) anneals to sequences in the constant regions of BCR cDNAs to amplify the entire BCR V(D)J region. During the PCR2, the nested hBCR LC (light chain) or hBCR HC (heavy chain) PCR2 Reverse primers anneal to sequences in BCR constant regions that are internal to the sequences bound by the hBCR PCR1 Reverse primer and add the Illumina Read 1 sequence (light purple). In the same PCR2 reaction, Unique Dual Index Kit primers anneal to the sequence added by hBCR PCR1 Universal Forward primer or the hBCR LC or HC PCR2 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 BCR light chain and/or heavy chain variable regions 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.