Illumina HiSeq 2500 paired end sequencing; Single-cell RNA-seq of of human dermal fibroblasts (supplementary - different stimulation conditions of dsRNA)
Single-cell RNA-seq of of human dermal fibroblasts (supplementary - different stimulation conditions of dsRNA)
Cells were sorted with Becton Dickinson INFLUX into wells containing 2uL of Lysis Buffer (1:20 solution of RNase Inhibitor (Clontech, Cat Number 2313A) in 0.2% v/v Triton X-100 (Sigma-Aldrich, Cat Number T9284), spun down and immediately frozen at -80°. Cell were grown in ATCC fibroblast growth medium (Fibroblast Basal Medium (ATCC, ATCC-PCS-201-030) with Fibroblast Growth Kit-Low serum (ATCC, PCS-201-041)  Cells were grown in three parallel time courses - each treated with 1ug/mL dsRNA (poly I:C) and LF-2,000. In one time course the poly I:C was replaced by fresh medium after 30mins, in a second time course the poly I:C was replaced after 1 hour; and in the third it was kept in the medium throughout the time course.  Cells were then collected after 2,4 and 6 hours. 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).
EMBL - EBI & Wellcome Trust Sanger Institute
E-MTAB-7052:25376_8_129_p