Name: GSM6547491
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: For circRNA-sequencing, RNA was extracted with Trizol. For FLASH-sequencing, the HEK293T_HNRNPD_3FHBH cells were crosslinked by UV irradiation. After a series of operations (harvested, lysed, sonicated, and immunoprecipitated), the cells were fixed with 90% ethanol and linked up with azide-labeled biotin in the click reaction solution (DPBS, 2.5 mM biotin-azide, 3 mM CuSO4, 6 mM Tris(3-hydroxypropyltriazolymethyl) amine, 10 mM aminoguanidine and 50 mM sodium L-ascorbate). Then the cells were washed with DPBS (containing 0.5% Triton X-100 and 2 mM EDTA) and DPBS. Discarding the DPBS, the cells were transferred into a 1.5 ml tube and suspended with lysis buffer (20 mM Tris-Cl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 0.5% lithium-dodecylsulfate, 5 mM DTT, and 0.1 U/µl RNase inhibitor) on the ice and homogenized through a syringe with a 0.4 mm diameter-needle 5 times. The nascent RNAs were immunoprecipitated with streptavidin-conjugated magnetic beads. Finally, the magnetic beads were washed with washing buffer and isolated from magnetic beads by RNA elution buffer (10 mM EDTA (pH 8.2) and 95% formamide), and digested with proteinase K. The nascent RNAs were extracted with Trizol reagent. For circRNA detection, samples were digested by RNase R to remove linear transcripts after ribosomal RNA (rRNA) depletion. Strand-specific libraries (also known as stranded/directional libraries) were prepared. For FLASH-sequencing, the immunoprecipitated RNA-HNRNPD complex was digested by RNaseI and TURBO DNase, followed by dephosphorylation. The complex was ligated with an s-oligo using T4 RNA ligase I, which was also followed by dephosphorylation. The complex was resuspended with proteinase K digestion buffer to release crosslinked RNA, and column purification (Zymo DNA Clean and Concentrator). The purified RNA was executed reverse transcription with SuperScript III, and column purification. The cDNA was then circularized with CircLigase. The cyclized cDNA was the template of PCR reaction with NEBNext High-Fidelity Master Mix. Then the PCR products were cleaned up with l.5 X AM pure beads. For RIP-sequencing, the Illumina Ribo-Zero Gold kit removed rRNA, and surplus RNA was purified for end repair and 50-adaptor ligation. Reverse transcription was performed with random primers containing 3' adaptor sequences. Finally, the cDNAs were purified and amplified with PCR reactions. The products with lengths of 300-500 were purified and quantified. These libraries were utilized for 150 nt paired-end sequencing with an Illumina Nova seq 6000 system (Novogene, China).