Bulk T cell receptor sequencing of Mus musculus: sorted CD8 IELs from small intestine of germ-free adult
TCR-alpha and TCR-beta chains from bulk sorted IELs were amplified using a 5 Rapid Amplification of cDNA Ends (RACE) with unique molecular identifiers (UMIs) for error correction essentially as described by Egorov et al., 2015. Briefly, RNA was isolated from sorted cell subsets using the RNeasy Micro Kit (Qiagen), and all the purified RNA was used to generate cDNA with SmartScribe RT, the SmartNNNa 5 adaptor, and TRAC/TRBC-specific primers to target the TCRa and TCRb chains. TCR chains were amplified from the cDNA using nested primers specific for the SmartNNNa adaptor and TRAC/TRBC during the 1st round PCR using Q5 Polymerase (New England Biolabs). The 2nd round PCR introduces unique 5 nucleotide sample-level barcodes attached to another set of nested primers specific for TRAC/TRBC used to distinguish CD8-, CD4-, and DN-IEL subsamples with the same index sequence. Barcoded TCR and TCR amplicons generated by the second round PCR were pooled by equal volume, prepped, and indexed for sequencing on Illumina platforms using a KAPA HyperPrep Kit (Roche).