Illumina HiSeq 2500 paired end sequencing; Single-cell RNA-seq of of dermal fibroblasts (supplementary - human fibroblasts stimulation with dsRNA)
Single-cell RNA-seq of of dermal fibroblasts (supplementary - human fibroblasts stimulation with dsRNA)
Cells were washed with PBS and then trypsinized.  Cells were grown in DMEM, high glucose, GlutaMAX Supplement, pyruvate (Life Technologies / 10569-010) + Fetal Bovine Serum (Lab Tech / FB-1001) + Penicillin – Streptomycin (Life Technologies / 15140122).  Cells were stimulated with 0.5ug/mL of  High-Molecular Weight poly I:C (Invivogen, Cat. Code: tlrl-pic), transfected with 1uL/mL Lipofectamin 2,000, for 2 or 6 hours before collection. Sorted plates were processed according to the Smart-seq2 protocol(Picelli et al., 2014): Oligo-dT primer (IDT), dNTPs (ThermoFisher, Cat Number 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, Cat Number 4456740) were added to each well, and Reverse Transcription (using 50U SmartScribe, Clontech Cat Number 639538) and PCR were performed following the original protocol with 25 PCR cycles. cDNA libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, Cat Number FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). Quality Checks on cDNA were done using a Bioanalyser 2100 (Agilent Technologies).  Libraries from 96 single cells were quantified using the LightCycler 480 (Roche), pooled and purified using AMPure XP beads (Beckman Coulter) with Hamilton 384 head robot (Hamilton Robotics). Pooled samples were sequenced on an Illumina HiSeq 2500 instrument, using paired-end 125-bp reads.