title: 454 GS FLX Titanium sequencing; High-throughput sequencing of IgH gene in minor salivary glands from Sjögren's syndrome patients reveals dynamic ectopic lymphoid structures with recirculating B cells
design: High-throughput sequencing of IgH gene in minor salivary glands from Sjögren's syndrome patients reveals dynamic ectopic lymphoid structures with recirculating B cells
library_construction_protocol: All labial salivary glands biopsies were obtained for diagnostic purposes, as approved by the National Research Ethics Service Committee London-Westminster (LREC 05/O0702/1), from patients attending the Rheumatology/Oral Medicine Sjögren's Clinic at Barts Health NHS Trust (London, UK). Total RNA was extracted from minor salivary glands tissue using the RNeasy Mini Kit (Qiagen); the concentration/purity of RNA samples were measured using the Nanodrop 20C (Lab Tech, UK) and the quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, UK).  To enrich for the Ig-VH genes, cDNA was synthesized using specific primers designed on the constant Ig-mu, -gamma and -alpha domains (Tiller et al 2008, Corsiero et al 2018) using 100 ng of total RNA and the Superscript III Reverse Transcriptase (Invitrogen) following the manufacturer's instructions. Thirty-six amplicon libraries were generated per sample, using the whole retro-transcribed RNA, 12 VH consensus forward primers, 3 Ig-mu, -gamma and -alpha reverse primers (Tiller et al 2008, Corsiero et al 2018) modified with a unique multiplex identifier (MID) tag and the Q5 High Fidelity DNA polymerase (NEB) according to the following conditions: a denaturation  at 98°C for 1' followed by 33 cycles (for gamma-mu isotypes) or 36 cycles (for -alpha isotype) of 98°C for 30'' and 72°C for 45'' followed by a final extension of 72°C for 2'.