GSM9438240: mESC_Mettl3cKO_4OHT_noAb; Mus musculus; RNA-Seq
Cells were fixed with 1.5% paraformaldehyde for 10 minutes, quenched with 125 mM glycine at room temperature for 5 minutes, and permeabilized with 0.5% Triton X-100 on ice for 10 minutes. Samples were then blocked with 1 mg/ml BSA, followed by sequential staining with a primary antibody, a secondary antibody, and protein A/G-reverse transcriptase. In situ reverse transcription was performed at 37°C for 1 h. Subsequently, cells were digested with proteinase K at 37°C for 2 h. Total nucleic acids were extracted using phenol-chloroform and concentrated by ethanol precipitation. Biotinylated cDNA was enriched using streptavidin beads. A 3' cDNA adapter (5′Phos-NNNNNNNNAGATCGGAAGAGCGTCGTGT-3′SpC3) was ligated using T4 RNA ligase 1 by incubating at 25°C for 16 h. The cDNA was then recovered from the beads. The library was prepared through PCR amplification using NGS sequencing primers, followed by gel purification to isolate fragments between 180 bp and 400 bp.