GSM2610097: miRNA_DC_31_S55_RACE-seq; Homo sapiens; OTHER
Monocytes: Peripheral blood monocytes were separated by leukapheresis of healthy donors, followed by density gradient centrifugation over Ficoll/Hypaque and subsequent counter current centrifugal elutriation in a J6M-E centrifuge. macrophages: Macrophages were generated by culturing monocytes in endotoxin-free RPMI 1640 medium (Biochrom) supplemented with 2% human pooled AB-group serum on Teflon foils for up to 7 days DCs: To generate immature DCs, 1 × 10^6 monocytes/ml were cultured in RPMI 1640 medium (Thermo Scientific, Bonn, Germany) supplemented with 10% FCS (Biowhittaker, Verviers, Belgium), 20 U/ml IL-4 (Promokine, Heidelberg, Germany) and 280 U/ml granulocyte-macrophage colony-stimulating factor (Berlex, Seattle, WA, USA).  GmbH, Wiesbaden, Germany) and cultured on teflon foils. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen) We mixed 4.0 μl 5X First-Strand Buffer, 0.5 μl DTT (100 mM; Invitrogen, catalog number 70726), 1.0 μl dNTP Mix (20 mM), spun briefly in a microcentrifuge, and kept at room temperature. We combined 1.0-10.0 μl with 1 μg total RNA from monocytes, macrophages, and dendritic cells, 1.0 μl Random Primer Mix (N-15) (20 μM), and 0-9 μl sterile water to reach a total volume of 11.0 μl in separate microcentrifuge tubes, mixed the contents and spun the tubes briefly. We incubated these tubes at 72 °C for 3 minutes, and then cooled to 42 °C for 2 minutes. After cooling, we spun the tubes for 10 seconds at 14,000 g to collect the contents at the bottom. Next, we added 1.0 μl of Smarter oligo (20 μM) per reaction, and mixed well by vortexing and spun the tube briefly in a microcentrifuge. We then added 0.5 μl RNase Inhibitor (40 U/μl; Invitrogen RNaseOUT™, catalog number 10777019) and 2.0 μl SMARTScribe Reverse Transcriptase (100 U; Clontech, catalog number 639537) to the buffer mix, and mixed these reagents at room temperature. Next, we added 8.0 μl of the master mix to the RNA solution, mixed the contents of the tubes by gently pipetting, and spun the tubes briefly. We incubated the tubes at 42 °C for 90 minutes and heated the tubes at 70 °C for 10 minutes in a hot-lid thermal cycler. We then added 90 μl Tricine-EDTA buffer to each tube. We prepared the master mix for the first PCR by combining 2.5 μl of the cDNA solution, 5.0 μl 10X Advantage 2 PCR buffer (Clontech, catalog number 639207), 1.0 μl dNTP Mix (10mM each) 50X Advantage 2 Polymerase Mix (Clontech), 1.0 μl of the smarterRACE_forward primer at 10 pmol/μl, 1.0 μl of the miRNA-specific outer primer (Table S20) at 10 pmol/μl, and added PCR-grade water to reach a volume of 50 μl. We ran a 2-step PCR program consisting of 1 minute at 95 °C, 25 cycles of 30 seconds at 95 °C followed by 70 seconds at 68 °C, 7 minutes at 68 °C, and finishing at 8 °C. We diluted 5 μl of the primary PCR product into 245 μl of Tricine-EDTA buffer. We prepared the master mix for the second PCR by combining 5.0 μl of the product of the first PCR after dilution with 5 μl of the 10X Advantage 2 PCR buffer, 1.0 μl dNTP Mix (10 mM), 1.0 μl of 50X Advantage 2 Polymerase Mix (Clontech), 2.0 μl of the Nextera_i7 primer, 2.0 μl of the miRNA-specific inner primer (Table S20), and 34 μl of PCR-grade water. We ran a 2-step PCR program consisting of 1 minute at 95 °C, 20 cycles of 30 seconds at 95 °C followed by 70 seconds at 68 °C, 7 minutes at 68 °C, and finishing at 8 °C. We diluted 5 μl of the PCR product into 245 μl of Tricine-EDTA buffer. We prepared the master mix for the third PCR by combining 5.0 μl of the PCR product of the second PCR with 5.0 μl of 10X Advantage 2 PCR buffer, 1.0 μl dNTP mix (10 mM), 1.0 μl of 40X Advantage 2 Polymerase Mix (Clontech), 2.0 μl of the Nextera_i7 primer, 2.0 μl of the Nextera_i5 primer, and 34 μl of PCR-grade water. We purified by AMPure at a 1.8 ratio, checked 2 μl of the second PCR product on a Tapestation, kept the libraries at -20 °C until sequencing, and pooled the PCR products, each with a different barcode combination before paired-end sequencing on a MiSeq sequencer (Illumina).