GSM5584850: 293T-shCFIm25-IP1; Homo sapiens; RIP-Seq
For RNA-seq, total RNA was extracted with TRNzol reagent (DP424, Tiangen). For Ψ-RIP seq, total RNA was extracted with TRNzol reagent (DP424, Tiangen). mRNA was isolated from the total RNA using the Dynabeads mRNA Purification Kit (61006, Invitrogen) for two round of poly(A)+ RNA selection and then using the RiboMinus Transcriptome Isolation Kit (K155002, Invitrogen) for rRNA depletion. RNA was fragmented into 150-300 nt fragments and incubated with anti-Pseudouridine mAb (D347-3, MBL). Then the mixture was subjected to Dynabeads protein G binding. After washing, the bound RNA was eluted by competition with Pseudouridine (HY-113061, MCE). RNA was recovered with Hieff NGS RNA Cleaner (12602ES03, Yeasen). For CFIm25-RIP seq, flag-3C-HA-CFIm25 stable overexpression HEK293T cells were crosslinked in 0.5% formaldehyde containing PBS. After DNase I treated, sonicated, incubated with MonoRab Anti-DYKDDDDK Affinity Resin (L00766, GenScript) and extensive washed, RNA was eluted by Recombinant HRV 3C Protease (S3CP01, SinoBiological ) cleavage. RNA-seq libraries were generated using  NEBNext Ultra II RNA Library Prep Kit (E7770, NEB ). RIP-seq libraries were generated using SMARTer Stranded Total RNA-Seq Kit v2 -Pico Input Mammalian (Takara) following the manual.