title: GSM5516474: BMDM_IL4_IL13_TNFa_rep_5; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated using MagMax (biosystems #AM1830) following the manufacturer's procedure. Final elution 50uL. Total RNA was quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (ThermoFisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 48-channel Fragment Analyzer (Agilent), respectively. RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). At total RNA input of 250ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEXNext Multiplex Oligos for Illumina #E7600 downstream (all New England Biolabs). The only deviation from the manufacturer's protocol was the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification, instead of the recommended SPRIselect Beads. Final sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). mRNA libraries were also assessed for size distribution and adapter dimer presence (<0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 48-channel Fragment Analyzer (Agilent). All sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina).