Dynabeads myone streptavidin c1 beads

Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were used to bind the biotin-labelled RNA to the streptavidin of the beads. Then, 400 pmol of biotin-labelled 10–37 or 74–95 RNA was bound to 200 µg of magnetic beads in the presence of binding and washing buffer 2X (10 mM Tris/HCl pH 7.5, 1 mM EDTA and 2 M NaCl). After 1 hour under slow shaking, the samples were washed three times with binding and washing buffer 1X and suspended in the same buffer. In another tube, 160 µg of M3 yeast proteins, prepared as previously described, was mixed with 1.5X REMSA buffer (100 mM HEPES pH 7.3, 200 mM KCl, 10 mM MgCl₂ and 10 mM DTT), 2 µl of 100 mM DTT, 3 µl of tRNA (10 µg/µl) in DEPC water and 100 µg of pre-washed beads. After 1 hour under slow shaking, the unbound proteins were transferred into the tube containing the RNA:beads complexes and the mixture was slowly shaken for 1 hour. Subsequently, ultraviolet (UV) cross-linking was performed by placing the tube underneath the light bulb of a 254 nm UV light source. The RNA-protein complexes were irradiated for 10 minutes, the supernatant was collected and the beads were resuspended in 20 µl of 1X LDS and boiled for 5 minutes. Samples were electrophoresed in a 12% polyacrylamide SDS-PAGE.

HTGTS libraries were constructed as described (Dong et al., 2015 (link)). Briefly, genomic DNA of CH12F3 or its mutants were extracted after stimulation for 3 days. The genomic DNA was sonicated and amplified by LAM-PCR with 5′ Sμ biotin primer (5′-CAG​ACC​TGG​GAA​TGT​ATG​GT-3′). The Biotinylated products of PCR were captured by Dynabeads MyOne streptavidin C1 beads (Invitrogen), ligated with bridge adapters on-bead. The ligated products were amplified by second-PCR to add adaptor. Then, the products of PCR were blocking with endonuclease Afill to remove germline genomic DNA fragment. The third round PCR was performed to add Illumina Miseq-compatible adapters to conduct MiSeq sequencing. The HTGTS data were analyzed as described (Dong et al., 2015 (link); Panchakshari et al., 2018 (link)). Data were presented as mean ± SEM (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001).

The genome library preparation was performed by using the Ion Xpress^TM Plus Fragment Library Kit (Life Technologies, USA) following the manufacturer’s protocol, 4471989 Rev E. The sheared DNA was separated using a 2% agarose gel (Promega, USA) and fragments that corresponded to 200 bp of a sequencing run (approximately 330 bp) were manually excised. The DNA was purified using the QIAquick Gel Extraction Kit (Qiagen, Germany). The library quality and concentration were assessed and determined using the 2100 Bioanalyzer (Agilent, USA) and High Sensitivity DNA Kit (Agilent, USA). The sequencing template was prepared using the Ion OneTouch^TM 200 Template Kit V2 DL (Life Technologies, USA) according to the manufacturer’s protocol. The Ion Sphere Particles were enriched using Dynabeads MyOne Streptavidin C1 beads (Invitrogen, Life Technologies, USA). The efficiency of the enrichment process was assessed using the Ion Sphere Quality Control Kit (Life Technologies, USA). Genome sequencing was undertaken using the Ion Torrent PGM sequencer (Life Technologies, USA).

Five microliters of 10 mg/ml Dynabeads MyOne Streptavidin C1 beads (Invitrogen) per 25 μl sample volume were prepared in bulk exactly as described previously (39 (link)). The resulting beads were resuspended at a concentration of ∼2 μg/μl in Buffer TX (1X transcription buffer [20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA (pH 8.0)] supplemented with 0.1% Triton X-100), split into in 25 μl aliquots, and stored on ice until use.