GSM5553057: Daoy_16_c: circRNA-SHH-4; Homo sapiens; ncRNA-Seq
Total RNA from cell lines or the medulloblastoma tumor sample was isolated with E.Z.N.A total RNA kit I (R6834-02) or total RNA midi kit (R6664-02) (Omega Bio-tek, USA) according to the manufacturer's instructions. On-column DNA digestion was performed with 40 KU (R6834-02 kit) and 60 KU (R6664-02 kit) of DNase (79254, Qiagen, Germany). a) Poly(A) enriched fraction: ≈ 100 ng of total RNA was used to generate stranded mRNA libraries with the TruSeq®Stranded mRNA Low Throughput Sample Preparation kit according to the manufacturer's instructions (15031047, Illumina, USA). This protocol involves a poly(A) selection/enrichment and ribosomal RNA depletion step. b) circRNA enriched fraction: 5 μg of total RNA was used. First, ribosomal RNA was depleted using the RiboMinusTM Eukaryote System V2 (Ambion) (A15026, ThermoFisher USA), followed by digestion with 5U RNase R for 1 hr to degrade linear RNAs and purification with the RNeasy MinElute Cleanup Kit (74204, Qiagen). The concentration and quality of the enriched circRNA RiboMinus/RNase R preparations were assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). 4.4 ng (for Daoy cells) or 1 ng (for HEPM cells) of RiboMinus/RNase R treated RNA was used for cDNA library generation. Random hexamer primers and Superscript III reverse transcriptase (18080055, Invitrogen, USA) were used for 1st strand synthesis and stranded mRNA library was generated according to the TruSeq®Stranded mRNA Low Throughput Sample Preparation protocol (15031047, Illumina, USA) as described above. The concentration and quality of the cDNA libraries were assessed with Qubit (Life Technologies, USA) and TapeStation, respectively. The cDNA libraries were normalized and pooled for sequencing on the Illumina NextSeq 550 platform using the Illumina NextSeq 500/550 High Output v2 kit (75 cycles; single read).