Spin column rna cleanup concentration kit

LwCas13a protein and its corresponding buffer were purchased from Magigen Biotech Co. Ltd. (Guangzhou, China). DNase I, T7 RNA polymerase, and recombinant RNase inhibitor were obtained from TaKaRa (Dalian, China). ChamQ Universal SYBR qPCR Master Mix was purchased from Vazyme (Guangzhou, China). Sangon Biotech (Shanghai, China) provided the NTP mixture (10 mM each), dNTP mixture (25 mM each), and Spin Column RNA Cleanup & Concentration Kit. DNA Constant Temperature Rapid Amplification Kit (Liquid Basic Type) and CRISPR Cas12/13 Hybridetect Test Note were purchased from Warbio Biotech (Nanjing, China).

SpyCLIP was performed according to a previous study^{28 (link)} with several modifications. Briefly, HEYA8 cell lines transfected with doxycycline-inducible SpyTag-FLAG-BUD31 expression lentivirus were induced with doxycycline for 72 h before harvesting. Cells were crosslinked and irradiated at 400 mJ/cm² in a UV Crosslinker (UVP, CL-1000). Cell nuclei were isolated with a Nucleoprotein Extraction Kit (Sangon) before lysis. Turbo DNase (2 U/μl, Invitrogen, AM2238) and 1:200 diluted RNase I (100 U/μl, Invitrogen, AM2295) were used to remove the DNA and to fragment the RNA. The mixed lysate was incubated with anti-FLAG magnetic beads (MBL, M185-11) at 25 °C for 40 min. After removing RNA-binding proteins from the FLAG beads with phosphoserine phosphatase, the mixture was incubated with fresh pre-washed SpyCatcher beads at 25 °C for 1 h. Stringent washes were conducted according to the manufacturer’s instructions, and the beads were digested with proteinase K (Roche, 3115828001). RNA was purified and concentrated with the Spin Column RNA Cleanup & Concentration Kit (Sangon), and the sequencing library was constructed with the NEBNext Ultra RNA Library Prep Kit for Illumina. High-throughput sequencing of the SpyCLIP libraries was performed on a HiSeq 2500 using the PE150 sequencing strategy (Novogene).