GSM7065995: E.HepG2.1k.PTBP1.rep1; Homo sapiens; OTHER
Cells were fixed with 1.5 % formaldehyde 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. Samples were blocked with 1 mg/ml BSA, followed by staining with primary antibody, secondary antibody and protein A/G-reverse transcriptase. Then, the in-situ reverse transcription was preformed at 37C for 30min. RT-primer 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.