title: NextSeq 550 paired end sequencing; In vitro RIP-seq for Zfp207 in mouse embryonic stem cells
design: In vitro RIP-seq for Zfp207 in mouse embryonic stem cells
library_construction_protocol: mESC were obtained from American Type Culture Collection (ATCC, Rockville, MD). The cell line was mycoplasma free when periodically tested with Mycoplasmacheck (Eurofins Genomics). mESC were cultured in T75 flasks at 37 °C and 5 % CO2 atmosphere using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1/100 Penicillin/Streptomycin (P/S, Sigma) and 10 % fetal bovine serum (Hyclone, GE healthcare). Huh7 cells were maintained by splitting 1/6 three times a week, done by aspirating the medium, gently washing the cells with phosphate buffered saline (PBS, without Mg2+, Sigma) and detaching them with 2 mL of a trypsin-EDTA solution (0.05%, Sigma) for 3-5 min. Trypsin-EDTA was inactivated with a minimum of 10-fold surplus of culture medium before a cell fraction was passaged. Extracted total RNA was fragmeted at 95 degrees celsius in Tris-HCl 80 mM and MgCl2 8 mM final concentrations for 4 minutes and 30 seconds. After fragmentation, the RNA was cleaned using 1 volume of RNA CleanXP Ampure beads and 1 volume of isopropanol; after 2 80% Ethanol washes the RNA was eluted in half the volume of AMPure beads used. Upon cell harvest, 700 µL Qiazol (QIAGEN) was directly added onto the cells on ice and mixed. The cell extract was either stored at -80 °C or RNA extraction was continued immediately by adding 140 µL chloroform. This Qiazol/chloroform mixture was shaken for 30 sec and incubated at RT for 3 min, before centrifugation at 9.000 g for 5 min at 4 °C. The upper aqueous phase was transferred to a new tube and an equal volume of isopropanol was added. The tube was inverted 5 times followed by 10 min incubation at RT. The mixture was centrifuged at 9.000 g and 4 °C for 10 min and the supernatant discarded. The pellet was washed in 700 µL cold 70% ethanol and centrifuged at 15.000 g for 5 min at 4 °C. The supernatant was discarded entirely, and the pellet air-dried for 5 minutes, before resuspension in nuclease-free water. RNA concentration was determined by nanodrop (Nanodrop 2000c). RNA was DNase-treated using the Turbo DNase Kit (Thermo Fisher) for 30 min at 37 °C and the reaction was stopped by adding the inactivation reagent from the kit and incubation for 5 min at 37 °C (Thermo Fisher). In order to achieve higher purity, the RNA was EtOH-precipitated overnight using 1/10 of 3M sodium acetate (pH 5.2) and 1 µL GlycoBlue (Ambion), followed by washing twice with 700 µ