TCR Deep-Seq of Sjogren's Disease patients : PBMC-sorted CD4+ T cells
RNA was extracted and purified using the RNeasy MinElute kit (Qiagen, USA) according to the manufacturers instructions. RNA yields were measured using the Qubit high sensitivity (HS) assay kit (Invitrogen, USA). A median of 400 ng of RNA (range: 70 to 400 ng) was used for cDNA synthesis. A modified cDNA-based library preparation protocol (Mamedov, I.Z., et al., Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling. Front Immunol, 2013. 4: p. 456) was adopted to prepare NGS libraries suitable for TCR deep sequencing. First-strand cDNA was labeled with unique molecular identifier (UMI) barcodes using the SmartScribe RT kit (Takara Bio, USA). The products were purified using Qiaquick PCR purification kits (Qiagen, USA). Two-stage amplification of TCR chains was performed as described previously (Egorov, E.S., et al., Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers. J Immunol, 2015. 194(12): p. 6155-63).  Samples with similar concentrations were pooled together for indexing and library preparation using the NEBNext Ultra II DNA kit from Illumina (New England Biolabs). The adaptor-tagged DNA from pooled samples was subjected to size-selection, and excess adaptors were removed using KAPA Pure beads (Roche Sequencing solutions). Following a PCR-amplification step using Illumina universal index primers, TCR libraries were gel-purified, and their integrity verified using TapeStation (Agilent Technologies).
SUB14680403
P2_BetaPool_6