title: RNA-Seq of synovial tissue biopsies from individuals with rheumatoid arthritis from the STRAP randomised clinical trial
design: RNA-Seq of synovial tissue biopsies from individuals with rheumatoid arthritis from the STRAP randomised clinical trial
library_construction_protocol: Patients underwent a synovial biopsy of a clinically active joint (clinically swollen and with ultrasound synovial thickening ≥2) before starting trial therapy. For the full study protocol and baseline patient characteristics see Rivellese et al., 2023 https://www.thelancet.com/journals/lanrhe/article/PIIS2665-9913(23)00241-2/fulltext Image analysis and base calling were conducted by the NovaSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de- multiplexed using Illumina's bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification. A minimum of 6 synovial samples per patient were immediately immersed in RNA-Later and RNA was extracted from synovial tissue using Qiagen Micro AllPrep RNA/DNA extraction kit. Briefly, 1-10mg of tissue was lysed using RLT buffer with addition of β-Mercaptoethanol, and then sheared using Qiagen TissueLyser II machine.Tissue lysate was then run through the kit columns set. Columns were washed using the appropriate kit wash buffers before RNA was eluted and re-suspended in RNAse free water. For more details see: https://www.thelancet.com/cms/10.1016/S2665-9913(23)00241-2/attachment/94785fe7-25eb-4792-8ea3-501537b9c5bc/mmc1.pdf RNA sequencing library preparation used the NEBNext Ultra II RNA Library Prep Kit for Illumina followed by manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNA were first enriched with Oligod(T) beads. Enriched mRNAs were 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 the Agilent 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA).