title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of of dermal fibroblasts
design: RNA-seq of of dermal fibroblasts
library_name: RH3_PIC4_p
library_construction_protocol: Cells were washed with PBS and then lysed in RLT plus buffer (Qiagen) 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 either: (1) 1ug/mL High-Molecular Weight poly I:C (Invivogen, Cat. Code: tlrl-pic) transfected with 2uL/mL Lipofectamin 2,000 (ThermoFisher, Cat Number 11668027); (2) mock transfected with Lipofectamin 2,000; (3) stimulated with 1,000 IU of IFNB (human IFNB - 11410-2 (for human and macaque cells); rat IFNB - 13400-1; mouse IFNB – 12401-1; all IFNs were obtained from PBL, and had activity units based on similar virological assays); or (4) left untreated. Additional samples in human and mouse were stimulated with 1,000 IU of Cross-mammalian IFN (Universal Type I IFN Alpha, PBL, Cat Number 11200-1).  Total RNA was extracted using the RNeasy Plus Mini kit(Qiagen, Cat Number  74136), using QIAcube (Qiagen).  Libraries were produced using the Kapa Stranded mRNA-seq Kit (Kapa Biosystems, Kit code: KK8421). The Kapa library construction protocol was modified for automated library preparation by Bravo (Agilent Technologies). cDNA was amplified in 13 PCR cycles, and purified by Ampure XP beads (Beckman Coulter, Cat Number A63882) (1.8x volume) using Zephyr (Perkin Elmer). Prior to sequencing, libraries were quantified using MiSeq (25bp PE). This data was used for pooling libraries in equimolar amounts by Coulter NX (Span-8, Beckman).
attributes.tag: Experimental Factor: organism
attributes.value: Macaca mulatta
attributes.tag: Experimental Factor: stimulus
attributes.value: poly I:C
attributes.tag: Experimental Factor: time
attributes.value: 4