GSM6048701: iCLIP-seq of wild-type SAMD4A-EGFP, replicate 1; Homo sapiens; RIP-Seq
Cells were UV cross-linked with 0.15 J/cm2 at 254 nm in a Stratalinker 1800 after induction with doxycycline (1 μg/ml) for 24 hours. Cells were lysed in 2.2 ml of iCLIP lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitors. To digest the genomic DNA and obtain RNA fragments of an optimal size range, 1 ml of lysate was incubated with Turbo DNase (Thermo Fisher Scientific, AM2238) and RNase I (1:250; Thermo Fisher Scientific, AM2294) for exactly 5 minutes at 37°C. EGFP-tagged proteins were immunoprecipitated using 5 μg of anti-GFP antibody (Abcam, ab290). A total of 2% input material was set aside for size-matched control libraries (SMI) prior to immunoprecipitation. Following three stringent washes with iCLIP high salt buffer (50 mM Tris–HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) and dephosphorylation with FastAP and T4 polynucleotide kinase, onbead-ligation of pre-adenylated adaptors to the 3' ends of RNAs was performed using the enhanced CLIP ligation method. The immunoprecipitated RNA was 5'-end-labeled with 32P using T4 polynucleotide kinase (New England Biolabs, M0201L). The protein-RNA complexes from the IP and SMI were separated using 4–12% BisTris–PAGE and transferred to a nitrocellulose membrane (Protran). For the SMI, the membrane was cut to match the size of the IP material. RNA was recovered by digesting proteins using proteinase K (Thermo Fisher, 25530049) and subsequently reverse transcribed into cDNA using barcoded primers. The cDNA was size-selected (low: 70 to 85 nt, middle: 85 to 110 nt, and high: 110 to 180 nt) using 6% TBE-urea and circularized using CircLigase™ II ssDNA ligase to add the adaptor to the 5' end. To enhance the circularization, betaine in the CircLigase reaction was added at a final concentration of 1 M, and the reaction mixture was incubated for at least 2 hours at 60°C. Circularized cDNA was digested at the internal BamHI site for linearization, and PCR amplified using Phusion High-Fidelity PCR Master Mix (NEB, M0531S). The final PCR libraries were agarose gel purified on purification columns (Qiagen), and the eluted DNA was mixed at a ratio of 1:5:5 from the low, middle, and high fractions and submitted for sequencing on Illumina NextSeq 500 platform using a full High-Output v2.5 flow cell to generate single-end 51 nucleotide reads with 40M read depth per sample.