RNAseq of 2D cocultures : Fibroblasts and B-cells from healthy or systemic sclerosis origin
Total RNAs were extracted from fibroblasts using the NucleoSpin RNA Mini kit (Macherey-Nagel, Dren, Germany). RNA quality (RIN) and concentration were measured on Bioanalyzer 2100 using RNA 6000 pico kit or RNA 6000 nano kit (Agilent Technologies, Santa Clara, CA, USA). 19 to 50 ng of RNA were used for library preparation with the QuantSeq 3 mRNA-Seq Library Prep Kit FWD (Lexogen, Vienna, Austria) with UMI Second Strand Synthesis Module. Quality and concentration of cDNA libraries were measured on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), then all libraries were pooled equimolarly, and quality checked again. Sequencing was performed on a NovaSeq 6000 sequencing system (Illumina, San Diego, CA, USA) with 100 cycles for an objective of 20M reads per sample (using a single-end mode).