also discussed some alternative solutions to tailor the analytic
method for specific usage. Several of the library preparation steps
have been optimized since the original protocol to make it more
efficient [21].2 Materials
All the materials used for RNA work should be RNase free. Com-
mon reagents and equipment are not listed here.2.1 Basic Reagents 1. PBS.
- S1 nuclease (e.g., ThermoFisher).
- 10 w/v % SDS (sodium dodecyl sulfate) in water.
- Proteinase K (e.g., Ambion).
- ShortCut RNase III (e.g., NEB).
- Gel loading dye, orange, 6.
- TRIzol LS reagent (e.g., Life Technologies).
- Chloroform.
- 100% ethanol.
- 80% ethanol.
- Isopropanol.
- 100% DMSO.
- 3 M Na-Acetate pH 5.5.
- Glycogen (e.g., GlycoBlue, 15 mg/mL).
- RNase inhibitor (e.g., Thermo Scientific SuperaseIn and
RiboLock). - T4 RNA ligase 1 (ssRNA ligase), high concentration (e.g.,
NEB). - RecJf exonuclease (e.g., NEB, cat. no. M0264).
- 5^0 Deadenylase (e.g., NEB, cat. no. M0331).
- Ultrapure TBE buffer, 10(e.g., Life Technologies).
- SequaGel UreaGel System, (National Diagnostics).
- Ultrapure TEMED (e.g., Invitrogen).
- Ammonium persulfate (APS).
- Reverse transcriptase (e.g., Invitrogen SuperScript III).
- 10 mM Deoxynucleotide solution mix (dNTPs).
- Magnetic streptavidin beads (e.g., Life Technologies Dyna-
beads MyOne streptavidin C1). - Magnetic SPRI beads (e.g., Beckman-Coulter AMPure XP).
PARIS: Psoralen Analysis of RNA Interactions and Structures 61