Illumina HiSeq 4000 sequencing; Bulk RNA sequencing of coding RNA from splenic dendritic cells isolated from interleukin-2 complex-treated and untreated mice
Bulk RNA sequencing of coding RNA from splenic dendritic cells isolated from interleukin-2 complex-treated and untreated mice
40,000 splenic mouse conventional dendritic cells (CD3-CD19-NK1.1-Ter119-CD45RB-CSFR1-MHC-II+CD11c+) from untreated and interleukin-2 complex treated mice were FACS sorted into RLT Plus lysis buffer (Qiagen) containing 1% 2-mercaptoethanol (Sigma-Aldrich). Interleukin-2 complex treatment consisted of 3 consecutive injections of 15'000 international units interleukin-2 (Teceleukin, Roche) mixed with 15µg NARA1 monoclonal antibody, as previously described (N. Arenas-Ramirez, C. Zou, S. Popp, D. Zingg, B. Brannetti, E. Wirth, T. Calzascia, J. Kovarik, L. Sommer, G. Zenke, J. Woytschak, C. H. Regnier, A. Katopodis, O. Boyman, Improved cancer immunotherapy by a CD25-mimobody conferring selectivity to human interleukin-2. Sci Transl Med 8, 367ra166 (2016)). RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) and stored at -80°C for approximately 3 weeks until further processing. Nucleic acid library construction was done by the Functional Genomics Center Zurich (University of Zurich, Swiss Federal Institute of Technology Zurich), as briefly described here: the RNA extracted from sorted cells was quantified for quality and concentration using the TapeStation RNA high sensitivity kit (Agilent). The estimated RINs were in a range of 8.4-9.8 showing a good RNA quality while the RNA concentrations of 1.1-10 ng/µl recommended the use of a low input library preparation method. Accordingly, the SMARTer Stranded Total RNA Seq Kit v2 (Takara Bio) was used to prepare cDNA by universal priming (with 3 min fragmentation) and to deplete ribosomal cDNA with ZapR v2 and R Probes v2. The Libraries were quantified by Tapestation D1000 (Agilent) measurements and they showed fragment sizes around 320bp.