title: GSM5027137: Donor_MM_4; Homo sapiens; RNA-Seq
library_construction_protocol: One million cells were collected, centrifuged, washed in PBS, and lysed in RLT buffer (79216, Qiagen) before storage at -80C. RNA was later extracted using the Qiagen RNeasy Mini Kit (74104, Qiagen, USA) according to manufacturer's specifications. An on-column DNA digestion (DNASE10-1SET, Sigma) was performed and the final elution volume was 30 uL. Total RNA was quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Q33140, ThermoFisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit (DNF-471-0500, Agilent) on a 48-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 25ng/μL while RIN ranged from 8.2 to 10, with a median of 9.9. Thirty-three human macrophage- and monocyte-derived RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). Total RNA input of 200ng 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. mRNA sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 16nM. Final library size distribution was also assessed (smear analysis of ~410bp average and adapter dimer presence <0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 48-channel Fragment Analyzer (Agilent). 32 out of 33 sequencing libraries passed quality check and were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot; and sequenced on a HiSeq 4000 Sequencing System (Illumina) with dual index, single read at 85 bp length (Read parameters: Rd1: 86, Rd2: 8, Rd3: 8), reaching an average depth of 24 million Pass-Filter reads per sample (14.6% CV).