title: NextSeq 550 paired end sequencing; RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and total purified cellular RNA
design: RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and total purified cellular RNA
library_construction_protocol: HepG2 were obtained from American Type Culture Collection (ATCC, Rockville, MD). The cell line was mycoplasma free when periodically tested with Mycoplasmacheck (Eurofins Genomics). HepG2 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). HepG2 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 Substrate preparation: DNAse treated and column cleaned 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. 3' dephosphorylation was then performed with T4PNK in TAM buffer (Tris-Acetate 50 mM, Magnesium Chloride 10 mM, pH~8.5) at room temperature for 4 hours followed by AMPure clean-up and subsequently 5' phosphorylated with T4PNK in T4PNK buffer with addition of ATP at 37 degreees celsius for 1 hour according to NEB manufacturer instructions, then AMPure cleaned and aliquoted and stored at -80 degrees celsius until further used. Binding Assay: RBP-Halo fusions are in vitro transcribed and then translated with MegaScript T7 in vitro transcription kit (ThermoFisher) and Wheat Germ extract according to manufacturers instructions (50 ul in vitro translation reaction). The in vitro translated proteins are recovered using 25 ul  Magne Halo Ligand beads (Promega) washed and resuspended in PBSN (1X PBS, 0,005% NP-40) with added RNAse A and Turbo DNAse (Thermo). After 1 hour of incubation, the beads are washed 4 times, twice with high salt (PBSN + 1M MgCl2) and twice with normal salt (PBSN). Beads are ruspended in 50 ul of PBSN and mixed with one aliquote of substrate RNA (150 ng in total) brought to room temperature and diluted to a final volume of 50 ul of PBSN. The binding reaction occurs at room temperature for one hour rotating end-over-end subsequently 3 washes in PBSN are performed to remove unbound molecules and elution is perfomed in 15 ul of RNAse free water at 70 degrees celsius for 8 minutes resuspending the beads by pipeting up and down whilst the tube is in the thermocycler for a total of 3 times during the 8 minutes at 70 degrees. 11 ul of the eluted RNA are used to prepare an illumina compatible next generation sequencing library.  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 column purified using RNA Clean&Concentrator kit (Zymo Research). The library preparation was carried out using the NEXTFLEX small RNA library preparation kit v3, PerkinElmer catalog # NOVA-5132-06 according to manufacturer instructions. The quality of every cDNA library was determined on an Agilent Bioanalyzer instrument according to the manufacturer's protocol.