12 channel fragment analyzer

FACS-sorted CD11b+CD11c+ and CD11b+CD11c− MDSCs were collected in RLT buffer (QIAGEN, Hilden, Germany) supplemented with 2-mercaptoethanol followed by RNA extraction using the RNeasy Mini Kit (Qiagen) and adding an on-column DNA digestion step according to manufacturer’s instructions. Total RNA was quantitatively and qualitatively assessed using the absorbance-based Take3 microvolume plate system on a Cytation 5 instrument (BioTek, Bad Friedrichshall, Germany) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 12-channel Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Concentrations averaged at 310 ng/µl while RIN values ranged from 8.6 to 10, with an average of 9.8.

MDSC-derived RNA samples were normalized, and a RNA input of 100 ng 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 (New England Biolabs, Frankfurt am Main, Germany). Ampure XP beads (Beckman Coulter, Brea, CA, USA) were used for double-stranded cDNA purification. mRNA sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher Scientific) on a Synergy HTX (BioTek). Library molarity averaged at 134 nM. Final library size distribution was assessed (smear analysis of 364 bp average and adapter dimer presence <0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 12-channel Fragment Analyzer (Agilent Technologies). All sequencing libraries passed quality check, were normalized, pooled, and spiked in with PhiX Control v3 (Illumina, San Diego, CA, USA). The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot and sequenced on a HiSeq 3000 Sequencing System (Illumina) with single index, single read at 85 bp length (Read parameters: Rd1: 85, Rd2: 8), reaching an average depth of 29 million Pass-Filter reads per sample (11% CV).