title: GSM5823390: 3: cells transfected with BPV-E4; Equus caballus; RNA-Seq
library_construction_protocol: The equine fibroblast cell lines were transfected using the nucleofection method with BPV-E4 and BPV-E4^E1 constructs. According to producers protocol, RNA was isolated from cell lines directly from culture dishes with PureLink™ RNA mini kit (Invitrogen, Thermo Scientific, Life Technology, USA). The procedure was maintained with the addition of the DNase treatment step (PureLink™ DNase Set, Invitrogen,Thermo Scientific, Life Technology, USA). The RNA was eluted with DEPC-Treated Water (Life Technologies, Ambion, USA). The quality and quantity of RNA were measured with 2200 TapeStation Automated Electrophoresis System (Agilent Technologies) as well as using nanodrop 2000 spectrophotometer (Thermo Scientific, Life Technology, USA) and agarose gel (2% agarose in TBE buffer) electrophoresis. All samples were sequenced using the NGS approach. According to the attached protocol, the high-quality RNA (RIN value from 9.3 to 9.8) was used for cDNA libraries preparation with theTruSeq RNA Kit v2 kit (Illumina, San Diego, CA, USA). The individual cDNA libraries were ligated with different indexes to pool samples during the NGS sequencing procedure. The quality and quantity of obtained libraries were assessed using Qubit 2.0 (Qubit™ dsDNA BR AssayKit, Invitrogen, Waltham, MA, USA ) and TapeStation 2200 (D100 screencaps, Agilent). Next, the cDNA libraries were sequenced on NextSeq 500 Illumina platform (Illumina, San Diego, CA, USA) and NextSeq 500/550 High Output KIT v 2.5 (75 cycles) according to protocol. The enrichment GeneOntology and over-represented Pathways analyses (KEGG, GO) were performed using David software (v6.8)  based on Equus caballus reference. The significance was shown based on False Discovery Rate (FDR)calculated as p-value after Benjamin multiple testing corrections (Benjamini and Hochberg, 1995). For visualization of gene interaction, String software [https://string-db.org/] was applied with Equus caballus reference. The cDNA libraries were prepared from 300ng of total RNA by using TruSeq RNA Kit v2 kit (Cat#FC-122-1001) according to protocol.