NextSeq 500 sequencing; Ago-RIP-Seq Analysis to Identify miR-375 Targets in Human Macrophages
Ago-RIP-Seq Analysis to Identify miR-375 Targets in Human Macrophages
Primary human macrophages from anonymous adult donors were provided as buffy coats were acquired from Blutspendedienst Frankfurt for PBMCs isolation. We used PBMCs from 6 donors (1 - 6) and the sample name indicates whether its a pool of donors 1-3 or 4-6. Macrophages were differentiated from PBMC using AB+ serum for 7 days. Macrophages were transfected with control or miR-375 overexpression mimic (MISSION® microRNA Mimic hsa-miR 375 from Sigma # HMI0537-5NMOL. For control tranfection we used MISSION® miRNA, Negative Control 2 Sequence from *C. elegans* also from Sigma #  HMC0003-5NMOL) for 48h then stimulated with MCF-7 apoptotic conditioning medium. After the stimulation, cells were washed and scrapped in PBS, centrifuged and pellet was lysed in PLB lysis buffer (5 mM MgCl2, 100 mM KCl, 10 mM Hepes, pH7.0 and 0.5% Nonidet P-40; freshly added  1 mM DTT, 100 U/ml RNase OUT (Invitrogen, Carlsbad, USA) and 1× complete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland)) and incubated on ice for 5 min Anti-pan-Ago IgG 2A8 (10 µg, Abcam) or IgG1 isotype control (ImmunoTools) were coupled to protein G Dynabeads (ThermoFisher Scientific) in 500 µL NT2 buffer overnight rotating at 4°C, followed by three washing steps with NT2 buffer. Thawed cell lysates were centrifuged for 30 min at 17,000g and 4°C and added 1:10 to NT2 buffer. The lysate was equally distributed to two tubes containing pan-Ago-IgG- or IgG1- coated beads and incubated overnight rotating at 4°C. The next day, beads were collected at the magnetic part and washed five times with 1 mL NT2 buffer. Protein-RNA complexes were eluted from the beads by incubation with 50 µL glycine (pH 2.3) for 15 min at room temperature and the obtained solution was neutralized using 5 µL Tris-HCL (pH 8.0). Proteins were digested by adding 3 µL of proteinase K (Sigma-Aldrich) and incubation for 10 min at 55°C. Thereafter, 350 µL PeqGold RNA Pure solution (Peqlab Biotechnologie) were added and RNA was purified using miRNeasy Mini Kit (Qiagen) without on-column DNase I digestion according to the manufacturers recommendations.  Purified RNA of Ago and IgG IP fractions was quantified using NanoDrop spectrometer and quality was assessed by Agilent capillary electrophoresis using RNA picoChips. 10 ng of RNA was DNaseI (Qiagen) treated and rRNA depleted by RiboZero (Epicentre). Then, sequencing libraries were generated with the SMARTer Stranded Total RNA Sample Prep Kit (Takara Clontech). Enrichment and size distribution of the libraries were quality-assessed by Agilent capillary electrophoresis on a DNA high sensitivity chip (Agilent). For sequencing, all 12 samples were pooled to generate a final concentration of 5nM per lane.
Experimental Factor: growth condition
non-conditioned media
Experimental Factor: stimulus
none
Experimental Factor: immunoprecipitate
anti-Ago2