New England Biolabs, E6330S, NEBNext® Immune Sequencing Kit (Mouse)

The large size of this product was discontinued on December 15, 2025. The small size will remain available. Related Categories Influenza Sequencing,, Next Generation Sequencing Library Preparation FAQ Q: What kind of samples can be used with the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)? A: RNA from a variety of sources can be used with the NEBNext Immune Sequencing Kit (Mouse), including: High quality RNA from B or T cells. The RNA should have a RIN score ≥7 to provide the highest percentage of full-length transcripts for library construction. When RNA is used with RIN scores <4, the complexity of the library is greatly reduced. RNA from any mouse sample that contains either B or T cells, such as peripheral blood PBMC or tissue. RNA extracted from frozen cell pellets, cultured cells, or cells sorted for specific B- or T-cell types. Q: Will the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330) work with non-mouse species? A: No, non-mouse primers (e.g., human) are not included in the mouse kit. For human primers, refer to NEBNext Immune Sequencing Kit (Human) (NEB #E6320). Q: Can I use FFPE tissue with the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)? A: It’s unlikely that FFPE tissue-derived RNA would be of sufficient quality to generate high-quality immune sequencing libraries. Q: What type of RNA extraction method should I use upstream of the NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)? A: Most common RNA extraction protocols should be sufficient, however we recommend the Monarch® Total RNA Miniprep Kit (NEB #T2010). Q: What sequencing parameters should be used with these libraries? A: Sequencing should be performed on an Illumina® MiSeq™, using Illumina V3 sequencing chemistry. We recommend running 300 bp x 300 bp, if possible (using a 600-cycle V3 MiSeq reagent kit), with the index read between the first and second reads. Q: What sequencing depth is required for immune sequencing libraries? A: The sequencing depth requirement is highly dependent on the biological questions that one is trying to answer. Typically, 500,000 reads per library is a good starting point for most projects to saturate a detection of 3000-4000 clonotypes. If the immune repertoire diversity of the RNA sample is higher, more sequencing reads are needed to detect all the low frequency clonotypes. Q: What barcodes should be sequenced together? A: The barcodes supplied with the kit are part of NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1, NEB #E7600). One can follow the product manual to choose barcodes for library pooling. Q: I don’t have a qPCR machine, can I still use NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)? A: The qPCR step is optional; it’s only used to identify the optimal final PCR cycling conditions, as each sample can contain varying amounts of amplifiable molecules. One can achieve a similar result by manually taking an aliquot of the PCR reaction every 5 cycles and visualizing each aliquot by diluting it 5-fold and loading on an Agilent Bioanalyzer® or TapeStation™ to identify the optimal cycling number that yields the highest product with the lowest background and overamplification. Typically, most samples will amplify best in the range of 6-18 cycles, depending on RNA input. Q: How do I analyze the data generated from immune sequencing libraries? A: There are several publicly available tools to perform data analysis. We recommend using the toolkit pRESTO, which is compatible with the NEBNext® Immune Sequencing Kit data, including unique molecular identifier (UMI) de-convolution. We have implemented the pRESTO pipeline on Galaxy for public use. Please visit this link for the pipeline and refer to our tutorial if you are new to the Galaxy platform. Q: Do I need to perform the qPCR step every time? A: It is best to perform the qPCR step every time a new RNA sample is used and/or a new type of library is prepared (e.g., B-cell receptor vs. T-cell receptor vs. both). When preparing a replicate library, the same PCR cycle number can be used as in the original prep. Q: Do I need to gel extract my final library? A: We have found gel extraction to be unnecessary for most libraries. A SPRI beads or AMPure® beads cleanup is usually sufficient at the end. Q: What B-cell gene and T-cell gene specific primers are included in NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330)? What is the format of the primers? A: NEBNext Immune Sequencing Kit (Mouse) includes primers for B-cell heavy chain and light chain (IGH/K/L), and primers for T-cell alpha chain, beta chain, gamma chain and delta chain (TRA/B/G/D). The primers included in this kit are provided in two tubes. One tube contains primers for B-cell heavy chain and light chain (IGH/K/L). The other tube contains primers for T-cell alpha chain and beta chain (TRA/B). Q: Will NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330) work with DNA? A: This kit is optimized to target RNA only. DNA can be present in the sample, but DNA will not be amplified by the primers included in the kit. Q: Will NEBNext Immune Sequencing Kit (Mouse) (NEB #E6330) provide heavy and light chain pairing information? A: No, this kit uses total RNA extracted from a desired sample; therefore, single cell information on pairing heavy and light chains will be lost.