GSM6758074: TCRseq from Pat14, blood --> PBMC --> T cell enriched; Homo sapiens; OTHER
4 mm punch biopsies were obtained from the same lesions sampled for metagenomics and at the same visit. The punch biopsies were immediately shock frozen and kept in liquid nitrogen until processed. RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (74704, Qiagen, Germany) along with the TissueLyser II (85300, Qiagen, Germany) according to the manufacturer's instruction. Peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn blood using density gradient centrifugation (Histopaque-1077, 10771, SIGMA-ALDRICH, Germany) and kept in liquid nitrogen until processed. PBMCs were thawed at 37° C, washed in pre-warmed RPMI 1640 medium (31870-025, Gibco, USA) including 10% FCS and counted using the Countess 3 machine (ThermoFisher Scientific, USA). To enrich the T cell population, the Pan T Cell Isolation Kit human (Miltenyi Biotec, Germany) was used according to the manufacturer's instructions. Directly thereafter total RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. Illumina sequencing libraries were constructed with the NEBNext Immune Sequencing Kit, human (E6320, New England Biolabs, USA) according to the manufacturer's instructions with 200 ng total RNA as starting material. The optimal number of PCR cycles for target amplification were determined for each sample by RT-qPCR as outlined in the manual. TCRseq, 300 bp PE using a 600-cycle V3 reagent kit