eCLIP sequencing library preparation

Enhanced crosslinking and immunoprecipitation (eCLIP) was performed on HepG2 cells in biological duplicates, essentially as described (41). For each replicate, extract from 20 × 106 UV-crosslinked (254 nm, 400 mJ/cm2) HepG2 cells was prepared by sonication in 1 ml lysis buffer, treated with RNase I (40 U, LifeTech), and immunoprecipitated overnight at 4°C with 2 μg affinity-purified rabbit anti-PKM2 antibody raised against a C-terminal peptide (Sigma cat. # SAB4200105, Lot #030M4874) pre-coupled to Dynabeads sheep M-280 anti-rabbit IgG beads (LifeTech). Prior to IP, a 20 μl aliquot of extract was removed and stored at 4°C for preparation of the size-matched input (SMInput) control. To ensure IP stringency, a series of wash steps was employed as follows: 2x low stringency wash buffer (20 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20), 2x high stringency buffer (15 mM Tris–HCl pH 7.4, 5mM EDTA, 2.5 mM EGTA, 1% Triton-X 100, 1% sodium deoxycholate, 0.1% SDS, 120 mM NaCl, 25 mM KCl), 2x high salt wash buffer (50 mM Tris–HCl, pH 7.4, 1M NaCl, 1mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), 2x low stringency wash buffer, and 2x PNK buffer (50 mM Tris–HCl pH 7.4, 10mM MgCl2, 0.5% NP-40). After washing, immunoprecipitated protein-RNA complexes were dephosphorylated and 3′-linker ligated on-bead to a custom oligonucleotide adapter. All samples (IPs and SMInputs) were heated in LDS Sample Buffer (LifeTech, 70°C, 10 min) and run on 4–12% NuPAGE polyacrylamide gels in MOPS running buffer (LifeTech). Complexes were wet-transferred to iBLOT nitrocellulose membranes in NuPAGE transfer buffer (all LifeTech) containing 10% methanol (overnight, 4°C, 30V). Immunoprecipitation was confirmed by performing standard immunoblotting on a fraction of all samples. RNA-protein complexes in the range from 75 kDa (the apparent molecular mass of PKM2) to 135 kDa (corresponding to PKM2-crosslinked RNAs of ∼220 nucleotides in length) were excised from the membrane. RNA was released by proteinase K treatment in urea and recovered by phenol chloroform extraction and column purification (RNA Clean-Up kit; Zymo Research). Input samples were dephosphorylated and 3′-linker ligated to a custom oligonucleotide primer and all samples (IPs and SMInputs) reverse transcribed using AffinityScript reverse transcriptase (Agilent). After treatment with ExoSAP-IT (Affymetrix) and alkali, cDNAs were recovered by purification on Dynabeads MyONE Silane beads (LifeTech), 5′-linker ligated on-bead to a custom oligonucleotide primer, purified, and recovered in 27 μl. cDNA was quantified by qPCR analysis of a fraction each sample using oligonucleotide primers specific to the 5′ and 3′ adapters. Half of the recovered cDNA was PCR-amplified (Q5 polymerase, NEB) using custom sequence-indexed oligonucleotide primers with the following cycle numbers: replicate 1: input 8, IP 15; replicate 2: input 8, IP 13. PCR products were purified (Agencourt AMPure XP beads; Beckman Coulter), size-selected to 175 -350 bp on 2% agarose gels, extracted (MinElute Gel Extraction kit; Qiagen), and quantified on a TapeStation using D1000 ScreenTape (Agilent). Libraries were sequenced on a HiSeq 4000 instrument (Illumina) in paired-end 55 bp mode. All sequence data was deposited to GEO (GSE229167).