title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of blood from donors with connective tissue diseases in the Lupus Extended Phenotype Cohort
design: RNA-seq of blood from donors with connective tissue diseases in the Lupus Extended Phenotype Cohort
library_construction_protocol: We have undertaken Lupus Extended Phenotype (LEAP) study in of a cohort of adult patients with connective tissue diseases (CTDs), namely systemic lupus erythematosus, Sjogren's syndrome, mixed and undifferentiated CTD, limited and diffuse cutaneous systemic sclerosis and dermatomyositis. RNAseq was undertaken in 12 participants from 4 'cohorts' based on interferon stimulated gene (ISG) and autoantibodies analyses: 3 ISG positive and anti-Smith positive participants; 3 ISG positive and anti-Smith negative participants; 3 ISG negative and anti-Smith positive participants and 3 ISG negative and anti-Smith negative participants. ISG measurements: Blood was collected into PAXgene tubes (PreAnalytix) and, after being kept at room temperature for between 2 and 72 hours, was frozen at -20oC, then stored at -80oC until extraction. Total RNA was extracted from whole blood using a PAXgene (PreAnalytix) RNA isolation kit. RNA concentration was assessed by Qubit 2.0 fluorometer (Thermo Fisher Scientific). After DNAse treatment, RNA was converted into cDNA with the High Capacity cDNA reverse transcriptase kit (Qiagen). Quantitative reverse transcription polymerase chain reaction (qPCR) analysis was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems), and cDNA derived from 40ng total RNA. Using TaqMan probes for IFI27 (Hs01086370_m1), IFI44L (Hs00199115_m1), IFIT1 (Hs00356631_g1), ISG15 (Hs00192713_m1), RSAD2 (Hs01057264_m1), and SIGLEC1 (Hs00988063_m1), the relative abundance of each target transcript was normalised to the expression level of HPRT1 (Hs03929096_g1) and 18S (Hs999999001_s1), and assessed with the Applied Biosystems StepOne Software v2.1 and Data Assist Software v.3.01. For each of the six probes, individual (patient and control) data were expressed relative to a single calibrator. The median fold change of the six ISGs, when compared to the median of the combined data of 29 healthy controls (16 female and 13 male) with a median age of 24 years (range 1-58 years), was used to create an interferon score for each patient as previously described (PMID: 24183309). RQ is equal to 2e-ddCt i.e. the normalized fold change relative to a control. The mean interferon score of the healthy controls plus two standard deviations above the mean (+ 2 SD) was calculated. Scores above this value (> 2•466) were designated as positive. Autoantibodies were measured in serum using the BioPlex 2200 ANA Screen with MDSS panel (according to the manufacturer's instructions). Total RNA was extracted from whole blood using a PAXgene (PreAnalytix) RNA isolation kit. RNA integrity was analyzed with Agilent 2100 Bioanalyzer.  Truseq stranded mRNA Assay. Paired end.
attributes.tag: Experimental Factor: clinical information
attributes.value: Smith autoantibody negative
attributes.tag: Experimental Factor: clinical history
attributes.value: interferon stimulated gene negative