title: Illumina HiSeq 2500 paired end sequencing; single-cell RNA-seq of of dermal fibroblasts
design: single-cell RNA-seq of of dermal fibroblasts
library_name: MM_pIC_2h_20_D8_p
library_construction_protocol: Cells were sorted with either 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°. Human dermal fibroblasts were obtained from the HipSci consortium (http://www.hipsci.org/ (Streeter et al., 2017) (where they were used for creating iPSCs). These original cells were thawed to test for growth and viability and refrozen after 1-2 passages, so that all lines were in the same growth rate and proliferation conditions before the stimulation experiment. Non-human primate cells were extracted from skin tissues that were incubated for 2hrs with 0.5% collagense-A (Roche; 11088815001) after mechanical processing, and then filtered through 100µm strainers before being plated and passaged prior to cryo-banking. Rodent cells were obtained from Cellbiologics where they were extracted using a similar enzymatic extraction protocol using skin tissues from the same regions of the body used in primates.  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 stimulated with 1ug/mL of rhodamine-labelled poly I:C (InvivoGen, tlrl-picr), transfected with 2uL/mL Lipofectamin 2,000  , for 0,2,4,8 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). Pooled samples were sequenced on an Illumina HiSeq 2500 instrument, using paired-end 125-bp reads.
attributes.tag: Experimental Factor: organism
attributes.value: Mus musculus