TCR-Seq for CD4 T cells from 2 C3H.HeSnJ mice to compare MHC context
Libraries were prepared and pre-processed as published (Ndifon et al. 2012). Briefly, we extracted total RNA from T cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA was then reverse transcribed using SuperScript II reverse transcriptase (RT) enzyme (Invitrogen, La Jolla, CA). The primer for the RT reaction was a TCR Cβ-specific primer linked to the 3'-end Illumina sequencing adapter. The resulting cDNA was then amplified using PCR (Phusion; Finnzymes) with a Cβ-3’adp primer and Vβ-specific 5’ primers. Each Vβ-specific primer was anchored to a restriction site sequence for the ACUI restriction enzyme. PCR products were then cleaned using QIAquick PCR purification kit (Qiagen, Hilden, Germany), followed by enzymatic digestion with ACUI enzyme (New England BioLabs, Ipswich, MA). Then, the 5’Illumina adaptor (dsDNA, with NN overhang) were ligated (T4 ligase; Fermentas, Vilnius, Lithuania). The adaptors also contained 3-nucleotide long tags for multiplexing of samples to the same Illumina sequencing run. A second round of PCR amplification was performed, using universal primers for the 5’ and 3’ Illumina adapters. Final PCR products were run on a 2% agarose gel, cut at the desired length (~250bp), and purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) to produce the final library. The libraries were sequenced using Genome Analyzer II or HiSeq2000 (Illumina).
Weizmann Institute of Science
TCR-Seq for CD4 T cells from 2 C3H.HeSnJ mice