Illumina HiSeq 4000 paired end sequencing; RNA-Seq of synovial tissue biopsies from individuals with rheumatoid arthritis from the R4RA randomised clinical trial
A minimum of 6 samples (ultrasound-guided synovial biopsies) per patient were immediately immersed in RNA-Later. All patients provided written informed consent before the synovial tissue biopsy procedure. They were fulfilling 2010 ACR/EULAR classification criteria for RA and were eligible for treatment with rituximab therapy according to UK NICE guidelines, i.e. failing or intolerant to csDMARD therapy and at least one biologic therapy (excluding trial IMPs) were recruited when fulfilling the trial inclusion/exclusion criteria (for the full study protocol and baseline patient characteristics see Humby et al). The patients were recruited under R4RA clinical trial.  For the full study protocol and baseline patient characteristics see Humby et al., 2021 https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)32341-2/fulltext#fig1 Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. Tissue was lysed in Trizol solution using a LabGen125 homogeniser. Then samples were extracted using Zymo Direct-zol Miniprep kits as per the manufacturer's instructions. Briefly, 1-10mg of tissue lysate was run through the Zymo-Spin IC Column. Columns were then washed using the appropriate kit wash buffers before RNA was eluted and re-suspended in RNAse free water. Quality control was carried out by quantifying samples by spectrophotometer readings on a Nanodrop ND2000C. RNA integrity was measured using Pico-chip technology on an Agilent 2100 Bioanalyzer to determine a RIN (RNA integrity number). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligo d(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 TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).