2015; 15:668C679. experiments. For tumorigenesis assay, A2780 cells were infected with shRNAs or bare vector, collected and resuspended in PBS. 5 106 cells in PBS were injected subcutaneously into one part of the posterior flanks of Balb/C nude mice at Isoshaftoside 6C8 weeks older. Tumor growth and volume were recognized every 3?days. After 4 weeks, mice were sacrificed and excess weight of xenografts was examined. For metastasis assays, 1 106 cells in PBS were injected into the abdominal cavity of three groups of Balb /c mice. After 4 weeks, mice were sacrificed and the numbers of metastatic nodules were counted. Gene-specific m6A qPCR and m6A sequencing (m6A-seq) Total RNAs were extracted and purified by using PolyTtract mRNA Isolation System (Promega, Hong Kong). After fragmentation, RNA was incubated with m6A antibody for immunoprecipitation according to the standard protocol of the Magna methylated RNA immunoprecipitation m6A Kit (Merck Millipore, Germany). Enrichment of m6A comprising mRNA was then analyzed either Isoshaftoside through RT-qPCR or high-throughput sequencing. Primers to m6A bad region of EEF1A was used as the bad control and primers to m6A postive region of EEF1A was used as the positive control according to the standard protocol of the Magna methylated RNA immunoprecipitation m6A Kit (Merck Millipore, Germany). For high-throughput sequencing, purified RNA fragments were Isoshaftoside used for library construction with the NEBNext Ultra RNA library Prep kit for Illumina (New England BioLabs) and were sequenced with Illumina HiSeq X Ten platform. Library preparation and high-throughput sequencing were performed by Novogene (Beijing, China). RNA sequencing (RNA-seq) RNA-seq was processed according to the instructions of NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs). Briefly, total RNAs were isolated from YTHDF1-depleted or control A2780 cells using Trizol reagent. Poly(A) RNA was consequently purified by using PolyTtract mRNA Isolation System and used to generate cDNA libraries. All samples were sequenced on Illumina HiSeq X Ten platform. Sequence reads were mapped to the human being genome version hg38 by using Illumina sequence analysis pipeline. The average gene expression ideals of three self-employed studies were used for following analysis. RNA immunoprecipitation and high-throughput sequencing (RIP-seq) Cells were washed twice with PBS, collected and then the pellet was resuspended in IP lysis buffer (150?mM KCl, 25?mM Tris (pH 7.4), Isoshaftoside 5?mM EDTA, 0.5?mM DTT, 0.5% NP40, 1 protease inhibitor, 1 U/l RNase inhibitor). The lysate was harvested by centrifugation at 12 000 g for 10 min after incubation for 30 min. Antibodies and 40 l of protein G beads (Invitrogen, USA) were added into the lysate followed by incubation over night at 4C. After washed three times with wash buffer (150?mM KCl, 25?mM Tris (pH 7.4), 5?mM EDTA, 0.5?mM DTT, 0.5% NP40), co-precipitated RNAs were extracted by Trizol reagent, ethanol-precipitated with glycogen (Invitrogen, USA). The enrichment of RNAs was normalized to IgG. For sequencing, Rabbit Polyclonal to p53 (phospho-Ser15) rRNAs was depleted by using the NEBNext rRNA depletion kit (New England BioLabs). cDNA libraries were produced by utilizing NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs) and sequenced on Illumina HiSeq X Ten platform. Each group was sequenced in duplicate. Enhanced UV crosslinking, immunoprecipitation and high-throughput sequencing (eCLIP-seq) eCLIP was performed as explained previously with small modifications (23). Briefly, cells were UV-crosslinked at 150 mJ and 254 nm wavelength in 10 cm plates with 10 ml of chilly PBS. Then cells were pelleted, flash freezing in liquid nitrogen, and stored at ?80C. The pellet was lysed with lysis buffer.