GSM6901140: YWHAH_rep1_INPUT; Homo sapiens; OTHER
All eCLIPs were conducted following induction or transient transfections and IP was conducted using anti-V5 tag (Bethyl A190-120A) and V5-tag or His-tag (both from Proteintech). eCLIP experiments were performed as previously described in a detailed standard operating procedure6 (6. Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508–514 (2016). ) In brief, 20 million cross-linked cells were lysed and sonicated, followed by treatment with RNase I (Thermo Fisher) to fragment RNA. Antibodies were precoupled to species-specific (anti-rabbit IgG) dynabeads (Thermo Fisher), added to lysate and incubated overnight at 4 °C. Before IP washes, 2% of sample was removed to serve as the paired-input sample. For IP samples, high-salt and low-salt washes were performed, after which RNA was dephosphorylated with FastAP (Thermo Fisher) and T4 PNK (NEB) at low pH, and a 3′ RNA adapter was ligated with T4 RNA ligase (NEB). Ten percent of IP and input samples were run on an analytical PAGE Bis-Tris protein gel, transferred to PVDF membrane, blocked in 5% dry milk in TBST, incubated with the same primary antibody used for IP (typically at 1:4,000 dilution), washed, incubated with secondary horseradish peroxidase-conjugated species-specific TrueBlot antibody (Rockland) and visualized with standard enhanced chemiluminescence imaging to validate successful IP. Ninety percent of IP and input samples were run on an analytical PAGE Bis-Tris protein gel and transferred to nitrocellulose membranes, after which the region from the protein size to 75 kDa above protein size was excised from the membrane, treated with proteinase K (NEB) to release RNA and concentrated by column purification (Zymo). RNA was isolated from the membrane, reverse transcribed with AffinityScript (Agilent), free primers were removed (ExoSap-IT, Affymetrix), and a 5' DNA adapter was ligated onto the cDNA product with T4 RNA ligase (NEB). Libraries were then amplified with Q5 PCR mix (NEB), size selected using AMPure XP beads (Beckman Coulter, Inc.) and on a 3% agarose gel, and then quantified with a Bioanalyzer instrument (Agilent). Paired-end (50 base pair) sequencing of the eCLIP libraries were performed on Illumina HiSeq 3500. enhanced CLIP