GSM5333021: ACHN_Input; Homo sapiens; RIP-Seq
Briefly, 1 x 107 cells that stably expressing Flag-tagged MEX3A were treated with 0.3% formaldehyde and the cross-linked reaction was stopped by adding 0.125 M glycine. The complexes of RNA and protein were enriched by 5μg of anti-Flag antibody with Dynabeads protein G (Invitrogen, #10004D) in cell lysis buffer overnight at 4 ºC. Then, total RNA was isolated by the phenol-chloroform method after treated with proteinase K (thermo fisher). Then, the library construction and sequencing were finished by BGI. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters.