Hiseq x ten sequencing system

Total RNA samples were extracted from rat bladder tissues using the Eastep Super Total RNA Extraction Kit (#LS1040; Promega) according to the manufacturer’s instructions. The profiling and quantitation of differentially expressed circular RNAs in rat bladder tissues were done via the RNA sequencing assay as previously described with minor modifications (Fu et al., 2017 (link)). Briefly, total RNA samples were incubated with DNase I (Promega), followed by removal of rRNA components using the Ribo-off rRNA Depletion Kit (#N406-01; Vazyme, Nanjing, and China) following the manufacturer’s instructions. Linear RNA molecules in total RNA samples were then deleted by treating with the RNase R. The resultant circular RNAs were fragmented and subjected to construction of RNA sequencing library via random priming using the Ultr II RNA Library Preparation Kit (#E7770S; NEB, United States) as instructed by the producer. Following end repair, dA-tailing, ligation of adaptors and uracil-specific excision, and the library was finally enriched by PCR method. After size selection, the library was then sequenced with an Illumina Hiseq X ten sequencing system.

Libraries were size selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification with a TBS380 fluorometer, the paired-end RNA-seq sequencing library was sequenced with an Illumina HiSeq X Ten sequencing system (2 × 150 bp read length). The data were analyzed using the free online Majorbio I-Sanger Cloud Platform (www.i-sanger.com).

Roots of control and treated seedlings were collected at 6 h and 15 d after treatment, immediately frozen in liquid N₂, and stored at −80 °C for further RNA-seq assays. Every RNA sample was derived from five independent seedlings. Total RNA was extracted from root samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) [56 ]. To eliminate genomic DNA contamination, the RNA samples were treated with RNase-free DNase I (Takara, Tokyo, Japan). The quality of the RNA was checked using an Agilent 2100 RNA Bioanalyzer (Agilent, Santa Clara, CA, USA). Libraries for RNA-Seq were prepared by using the VAHTS mRNA-Seq v2 Library Prep Kit (Vazyme Biotech Co., Ltd., Nanjing, China). Sequencing was performed using a HiSeqXTen sequencing system using stranded paired-end 150 bp sequencing (Illumina, Inc., San Diego, CA, USA).

m⁶A-MeRIP using low-input materials was performed based on a previously described protocol [55 (link)] with some modifications. Briefly, total RNA from about 2500 mouse GV oocytes from 5 weeks C57BL/6 J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) was first randomly fragmented to ~200 nt with RNA fragmentation reagents (AM8740, Thermo, USA), and then incubated with the protein A beads (10001D, Thermo, USA) coupled with anti-m⁶A polyclonal antibody (ABE572, Millipore, USA) in IPP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40, 0.4 U/μl RNasin). After immunoprecipitation, the RNA reaction mixture was washed twice in 1 ml of IP buffer, twice in 1 ml of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% IGEPAL^® CA-630 (I8896, Sigma-Aldrich, Germany) in nuclease-free H₂O), and twice in 1 ml of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% IGEPAL CA-630 in nuclease-free H₂O) for 5 min each at 4 °C. After extensive washing, the m⁶A-enriched RNA fragments were eluted from the beads by proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA was subjected to library construction using the SMARTer Stranded Total RNA-Seq Kit v2 (634413, Clontech, Japan) according to the manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq X-ten sequencing system.

RNA isolation and RNA‐Seq were performed as described previously.25 Briefly, 5 × 10⁶ ESCs were harvested and lysed using 1 mL TRIzol reagent (Invitrogen). The RNA was purified using 1:5 (v/v) chloroform (Sinopharm Chemical Reagent Beijing Co., Ltd) and precipitated by centrifugation using 1:1 (v/v) isopropanol alcohol (Sinopharm Chemical Reagent Beijing Co., Ltd). The RNA pellet was dissolved in RNase‐free ddH₂O (Invitrogen) at a concentration of about 1 μg/μL. The cDNA libraries were constructed using the TruSeq RNA Sample Prep Kit with 10 μg of the isolated RNA per sample. All samples were sequenced by an Illumina HiSeq‐X TEN sequencing system with paired‐end 150‐bp read length. For the data analysis, the mm10 version annotation for mouse genome were used. Clean RNA‐Seq reads were mapped by HISAT2 with default settings (version 2.1.0). Reads with unique genome location were used for splicing analysis. rMATS were used to identify differentially splicing events between wild‐type and Rbm14 knockout samples. The thresholds were set at D‐value of percent spliced in (ΔPSI) = 10% and false discovery rate (FDR) = 0.05. The sashimi plots were produced by rmats2sashimiplot (https://github.com/Xinglab/rmats2sashimiplot/). The gene ontology analysis was performed by DAVID (version 6.8), and the figures were plotted by ggplot2.