title: Illumina HiSeq 2500 sequencing; Transcription profiling by RNA sequencing of MelanA- and caIKK-treated monocyte-derived dendritic cells
design: Transcription profiling by RNA sequencing of MelanA- and caIKK-treated monocyte-derived dendritic cells
library_construction_protocol: Monocyte-derived DCs were generated as previously described in our previous study (PMID: 15728524). In brief, blood samples from seven healthy donors were collected after approval was granted by the responsible institutional review board (Ethikkommission der Friedrich-Alexander-Universität Erlangen-Nürnberg, Ref. no. 4158) and written informed consent was obtained. Peripheral blood mononuclear cells were isolated from whole blood using density gradient centrifugation. Monocytes were extracted from the non-adherent fraction by plastic adherence, were cultured in DC medium (RPMI (Lonza, Verviers, Belgium) containing 1% non-autologous human plasma (Sigma-Aldrich, St Louis, USA), 2 mM L glutamine (Lonza), and 20 mg/L gentamicin (Lonza), and were differentiated into DCs by application of 800 U/mL GM CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) and 250 IU/mL IL 4 (Miltenyi Biotec) on days 1, 3, and 5. After six days in culture, DCs were matured for 24 hours using a cytokine cocktail consisting of 200 IU/mL IL 1β (CellGenix, Freiburg, Germany), 1 000 U/mL IL 6 (CellGenix), 10 ng/mL TNF (Beromun, Boehringer Ingelheim Pharma, Germany), and 1 μg/mL PGE2 (Pfizer, Zurich, Switzerland). Generation of in vitro transcribed RNA with mMESSAGE mMACHINE™ T7 ULTRA transcription-kits (Thermofisher scientific, Waltham, USA) and the electroporation of cocktail-matured DCs (cmDCs) was carried out as previously described (PMID: 28717398). DCs electroporated with RNA encoding melanoma antigen recognized by T cells 1 (MelanA) were used as control. cmDCs were electroporated using 30 µg RNA encoding constitutively active IKKβ (caIKK) and such DCs were used as case.  Approx. 5,000 ng RNA including small RNAs was extracted 4 hours after electroporation using the RNeasy Plus Mini Kit (QIAGEN GmbH, Hilden, Germany). For each sample, 200 ng of total RNA was used to construct sequencing libraries using the RNeasy Plus Mini Kit, according to the manufacturer's protocol (QIAGEN GmbH, Hilden, Germany).