RNAseq of 3D cocultures : Fibroblasts from 3D human reconstitutes healthy skin cocultured with healthy or systemic sclerosis B-cells
Fibroblasts were purified from cocultures by FACS sorting and Total RNAs were extracted from fibroblasts using the NucleoSpin RNA Mini kit (Macherey-Nagel, Dren, Germany). Libraries were prepared with the following kits: NEXTflex Poly(A) Beads 2.0. for isolation of mRNAs, NEXTflex Rapid Directional RNA-seq Kit 2.0 for cDNA generation and library construction, and NEXTFlex Unique Dual Index Barcodes - Set A for indexation (PerkinElmer, Shelton, CT, USA). cDNA libraries were analyzed for length and concentration on Bioanalyzer 2100 with the DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA) and on Qubitflex 4 with the Qubit dsDNA BR assay kit (ThermoFisher Scientific, Waltham, MA, USA) respectively. Bulk RNAsequencing was performed on a NovaSeq 6000Dx sequencing system (Illumina, San Diego, CA, USA) in a 2x75bp configuration (using paired-end reads).