Illumina HiSeq 2500 paired end sequencing; Bulk RNA-seq of of human dermal fibroblasts from up to 66 individuals (supplementary)
Bulk RNA-seq of of human dermal fibroblasts from up to 66 individuals (supplementary)
Cells were washed with PBS and then lysed in RLT plus buffer (Qiagen) 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 0,2 or 6 hours before collection. 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).