GSM7730935: H.HepG2.40k.RBFOX2.noRNase.13nt.rep1; Homo sapiens; OTHER
Cells were fixed with 1.5 % paraformaldehyde for 10 minutes, quenched with 125 mM glycine at room temperature for 5 min, and permeabilized with 0.5% Triton X-100 on ice for 10min. For samples with RNase treatment, RNase I was added and samples were incubated at 37C for 5 min. Samples were blocked with 1 mg/ml BSA, followed by staining with primary antibody, secondary antibody and protein A/G-reverse transcriptase. For samples with strong RNase treatment, RNase I was added and samples were incubated at 37C for an additional 5 min. Then, the in-situ reverse transcription was preformed at 37C for 30min. Cells were digested with proteinase K at 37C for 2h, and total nucleic acids were extracted with phenol-chloroform extraction and concentrated by ethanol precipitation. Biotinylated cDNA were enriched by strepavidin beads. 3' cDNA adapter (5′Phos-NNNNNNNNAGATCGGAAGAGCGTCGTGT-3′SpC3) was ligated by T4 RNA ligase 1 by incubating at 25 °C for 16 h. cDNA was recovered from beads. Then, the library can be obtained by PCR amplification with NGS sequencing primer and gel purification of size between 180bp and 400bp. OTHER: ARTR-seq (an Assay of Reverse Transcription-based RBP binding sites Sequencing)