RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and purified total cellular RNA
RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and purified total cellular RNA
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 fragmented at 95°C 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 washing the RNA pellet twice with 80% Ethanol, RNA was eluted in half the volume of AMPure beads used. 3' dephosphorylation was then performed with T4-PNK 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 T4-PNK in T4-PNK buffer with addition of ATP at 37°C for 1 hour according to NEB manufacturer instructions, then AMPure cleaned and aliquoted and stored at -80°C until further used.   Binding Assay: RBP-Halo fusions are in vitro transcribed and then translated with MegaScript T7 in vitro transcription kit (Thermo Fisher Scientific) and Wheat Germ extract (Promega) according to manufacturers instructions (50 µL in vitro translation reaction). The in vitro translated proteins are recovered using 25 µL Magne Halo Ligand beads (Promega) washed and resuspended in PBSN (PBS, 0,005% NP-40) with added RNase A and Turbo DNase (Thermo Fisher Scientific). After 1 hour of incubation, the beads are washed 6 times, three times with high salt (PBSN + 1M MgCl2) and three times with normal salt (PBSN). Beads are resuspended in 50 µL of PBSN and mixed with one aliquot of substrate RNA (150 ng in total) brought to room temperature and diluted to a final volume of 50 µL of PBSN. The binding reaction occurs at room temperature for one hour rotating end-over-end subsequently three washes in PBSN are performed to remove unbound molecules and elution is performed in 15 µL of RNase-free water at 70°C for 8 minutes resuspending the beads by pipetting up and down while the tube is in the thermocycler for a total of 3 times during the 8 minutes at 70°C. A total of 11 µL of the eluted RNA are used to prepare an Illumina compatible next generation sequencing library.  For mod-RAPseq, a total of 2 ng of the RAPseq input library were PCR-amplified using primers F_T7_RAPseq and R_T7_RAPseq (see Supplementary Table 1) and Q5® DNA Polymerase (NEB) for 20 cycles. The PCR product was purified using AMPure XP beads (Beckham Coulter) at a 2X bead-to-sample ratio, following the manufacturer's protocol. Next, 500 ng of the purified PCR product was used as the template for in vitro transcription using the MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific). Transcription was performed at 37°C for 4 hours. The in vitro transcription reaction was then treated with 1 μL of TURBO™ DNase (Thermo Fisher Scientific) and incubated at 37°C for 1 hour to remove DNA templates. RNA was purified using RNA Clean XP beads (Beckham Coulter) at a 1.5X ratio per the manufacturer's instructions. The final RNA product was quantified using the QubitTM microRNA assay kit (Thermo Fisher Scientific), diluted to 50 ng/μL and stored in 3μL aliquots at -80°C. RNA affinity purification was performed as described. For reverse transcription, 11 μL of RAPseq eluate were mixed with 1 μL of 10 μM R_T7_RAPseq primers in a 20 μL reaction containing SSII (Thermo Fisher Scientific), and incubated at 42°C for 45 min. The reaction was heat-inactivated at 90°C for 10 min. cDNA was purified according to the NEXTFLEX Small RNA Library Preparation Kit cleanup procedure. Purified cDNA was then PCR-amplified in a 25 μL reaction using 0.5 μL Q5 DNA polymerase (NEB), 1 μL of universal and 1 μL of barcoded primer (both from the NEXTFLEX small RNA library preparation kit). After six cycles of amplification, the PCR product was purified using AMPure XP beads (Beckham Coulter) using a 1.5X bead-to-sample ratio.   For co-RAPseq, a total of 25 μL of Magne® HaloTag® Ligand beads (Promega) were washed three times with PBSN. After removing the storing solution, the beads were resuspended with 100 μL of a mixture containing two combined 50 μL in vitro translation reactions for HUR and PTBP1, respectively. RNA affinity purification and library preparation was performed as described in protocol 5 and 3.  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. 