title: RNA-sequencing from human salivary glands from Sjogren's disease patients recruited in the TRACTISS randomized clinical trial
design: RNA-sequencing from human salivary glands from Sjogren's disease patients recruited in the TRACTISS randomized clinical trial
library_construction_protocol: One LSG lobule per patient was stored in RNAlater solution for 24 hours at 4C and transferred at −80C for long-term storage. NA NA Patients received either two doses, two weeks apart, of intravenous rituximab (1,000 mg) or placebo (250 mL saline) in two courses at weeks 0 and 2, 24, and 26 in combination with corticosteroid in both arms. NA SG RNA was extracted usingRNeasy Micro Kit (Qiagen) as per the manufacturer's instructions, quantiﬁed and checked for integrity using Qubit-2.0 Fluorometer(Invitrogen) and TapeStation (Agilent Technologies), respectively. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Briefly, mRNA was first enriched with Oligod(T) beads and fragmented for 15 minutes at 94°C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on TapeStation and quantified with Qubit 2.0 Fluorometer and quantitative PCR (KAPA Biosystems).