Targeted TCR Amplification from Single-Cell cDNA Libraries
Single-cell sequencing of TCR alleles enables determination of T cell specificity. Here we describe a sensitive protocol for targeted amplification of TCR CDR3 regions from single-cell full-length cDNA libraries. By exploiting the specificity of RNase H-d
- PDF / 437,856 Bytes
- 28 Pages / 504.567 x 720 pts Page_size
- 22 Downloads / 187 Views
1
Introduction The diversity of individual T cell receptor (TCR) chains has been used as a measure of clonal diversity in the analysis of the immune response to pathogens and vaccines [1]. The recent successes of immunotherapy for cancer have intensified interest in TCR analysis because T cells are the primary effector cells of immune response to tumor [2]. Interaction of a TCR with a peptide antigen bound to a major histocompatibility complex (MHC) molecule occurs mainly through the paired alpha- and beta-CDR3 regions. Thus, determining the sequence of these CDR3 segments is a necessary component in characterizing the antigen specificity of a T cell. Identification of which TCR interacts with a particular antigen requires single-cell sequencing so the exact pairing of TCR-alpha and TCR-beta chains is known. Programs such as TraCeR [3] enable extraction of TCR sequences from single-cell RNA-seq data as long as the sequencing uses whole transcript libraries rather than end counting libraries. Due to read-depth limitations of single cell RNA-seq, this type of analysis does not always detect both TCR alpha and beta sequences in the same single cell. Also, it sometimes recovers only partial CDR3 sequences. Complete CDR3 sequence information is critical for the cloning and expression of TCRs, which is required for determining the specificity of discovered
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_13, © Springer Science+Business Media, LLC, part of Springer Nature 2019
197
198
Shuqiang Li and Kenneth J. Livak
TCRs. Thus, there is a need to improve the sensitivity of determining TCR sequences in single-cell cDNA libraries. Here we present a protocol that starts with full-length singlecell cDNA libraries suitable for preparing whole transcript sequencing libraries and achieves specific, targeted amplification of TCR alleles plus addition of cell barcodes in a single PCR step, as opposed to a nested PCR strategy such as the one described by Han et al. [4].
2
Materials 1. 96-Well PCR plates. 2. 20 mg/mL Proteinase K (800 units/mL). Store at
20 C.
3. Prionex: Sigma-Aldrich G0411. 4. NEBNext Single Cell/Low Input RNA Library Prep Kit: New England BioLabs, E6420L. 5. 25 mM AAPV protease inhibitor: Dissolve 5 mg Elastase Inhibitor III (Sigma-Aldritch 324745-5MG) in 400 μL DMSO. Dispense into 20 μL aliquots and store at 80 C. This is a peptide inhibitor with the amino acid sequence AAPV. 6. Thermal cycler. 7. ProNex beads: Promega NG2001(see Note 1). Product includes Promega Wash Buffer concentrate and Promega Elution Buffer. 8. Ethanol: Used to dilute Promega Wash Buffer concentrate and to prepare 80% ethanol. 9. Magnetic stand for 96-well plate. 10. DNA Suspension Buffer: 10 mM Tris–HCl, pH 8.0 and 0.1 mM EDTA. 11. Set of 96 barcode primer mixes: Each mix consists of 6 μM rhPCR primer P5.IDT***.Rd1x.x1 and 6 μM rhPCR primer P7.IDT***.Rd2x.x1, where IDT*** refers to the different barcodes. The sequence
Data Loading...
