title: GSM5896591: MK 1h rep2; Homo sapiens; Myxoma virus; RNA-Seq
library_construction_protocol: THP-1 cells (106 cells per 3.5 cm dish) were differentiated in phorbol myristic acid (PMA) (Millipore-Sigma, )at 50 ng/mL for 48 hours before mock treated, transfection with ISD at 2 mg per dish using ViaFect (Promega, Madison, WI) at 3 mL per 1 mg of ISD based on manufacturer recommendations, or infected at an moi of 5 for either WT (vMyxGFP) or M062R-null MYXV.  At 8- hour post-transfection of ISD and at 1- and 8- hour post-viral infection cells were harvested, spun down in cell pellets, and then stored in -80°C until RNA extraction. RNA was extracted using the Quick DNA/RNA Mini-Prep Plus kit (catalog # D7003; Zymo, Irvine, CA, USA) with on-column DNase digestion. Purified RNA was assessed for mass concentration using the Qubit RNA BR Assay kit (catalog # Q10211; Invitrogen, Waltham, MA, USA) and for integrity using the Standard Sensitivity RNA Analysis kit on a Fragment Analyzer capillary electrophoresis system (catalog # DNF-471-0500; Agilent, Santa Clara, CA, USA). A total of 250ng total RNA was used for each sample as input to the TruSeq Stranded Total RNA library prep kit with unique-dual indexing (catalog #s 20020598 & 2002371; Illumina, San Diego, CA, USA). Libraries were assessed for mass using the Qubit 1X dsDNA HS Assay kit (catalog # Q33231; Invitrogen, Waltham, MA, USA), for fragment size using the High Sensitivity NGS Fragment Analysis kit on a Fragment Analyzer capillary electrophoresis system (catalog # DNF-474-0500; Agilent, Santa Clara, CA, USA), and functional validation using the Universal Library Quantification kit (catalog # 07960140001; KAPA, Wilmington, MA, USA). Validated libraries were adjusted to 3nM before pooling, denaturing, and clustering. Paired-end (2X75) sequencing was performed to an average of 40 million reads per sample on a HiSeq 3000 (Illumina, San Diego, CA, USA).