title: Illumina HiSeq 4000 paired end sequencing; RNA-Seq of human aortic endothelial cells with lysophosphatidylcholine, IL-35, and IL-10 against controls
design: RNA-Seq of human aortic endothelial cells with lysophosphatidylcholine, IL-35, and IL-10 against controls
library_construction_protocol: HAECs were collected using RNAeasy kit. Human aortic endothelial cells (HAECs) (Lonza, CC2535; Walkersville, MD) were cultured in medium M199 (Hyclone laboratories, Logan, UT) supplemented with 15% fetal bovine serum (FBS; HyClone); endothelial cell growth supplement (ECGS, 50μg /mL; BD Biosciences, San Jose, CA); heparin (50μg/mL); and 1% penicillin, streptomycin, and amphotericin (PSA; Invitrogen, Carlsbad, CA).  HAECs were grown on 0.2% gelatin-coated flasks, plates, or dishes and experiments were performed at passage 9. 18-hour vehicle or lysophosphatidylcholine (16:0) treatment with or without interleukin 35 (IL-35)/IL-10 cotreatment RNA is extracted using RNeasy kit, then mRNA and non-coding RNAs are enriched by removing rRNA from the total RNA with kit.   By using the fragmentation buffer, the mRNAs are fragmented into short fragments (about 200~500nt), then the first-strand cDNA is synthesized by random hexamer-primer using the fragments as templates, and dTTP is substituted by dUTP during the synthesis of the second strand.  Short fragments are purified and resolved with elution buffer for end reparation and single nucleotide A (adenine) addition.  After that, the short fragments are connected with adapters, then the second strand is degraded finally using UNG(Uracil-N-Glycosylase).  After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates.  During the quality control steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.