Illumina HiSeq 2000 paired end sequencing; Differential Ago-RIP-Seq for the identification of miR-375 targets in prostate cancer cells
Differential Ago-RIP-Seq for the identification of miR-375 targets in prostate cancer cells
PC-3 cells were treated with pREP4_pre-miR-375 expression vector or pREP4 empty vector as a control using Extreme Gene HP (Roche) Purified RNA of TL, 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 six samples of one replicate of an IP experiment (TL, Ago-IP, IgG-IP fraction of PC-3 miR-375 and PC-3 vector, respectively) were pooled to generate a fincal concentration of 5nM per lane.
Experimental Factor: empty vector
genotype
Experimental Factor: none
immunoprecipitate