4
Library preparation
Approx 160 Mins
Goal: to use the Direct RNA Sequencing Kit to prepare an RNA control library.
Library preparation using the Direct RNA Sequencing Kit takes approximately 2 hours.
The protocol consists of three steps:
- Reverse transcription of the RNA control strand (S. cerevisiae enolase 2 transcript)
- Cleaning up the sample using RNAClean XP beads
- Ligation of sequencing adapters
What you will need
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- 10 mM dNTP solution (e.g. NEB N0447)
- Agencourt RNAClean XP beads (Beckman Coulter™, cat # A63987)
- Direct RNA Sequencing Kit (SQK-RNA004)
- Freshly prepared 70% ethanol in nuclease-free water
- Hula mixer (gentle rotator mixer)
- Ice bucket with ice
- Magnetic rack
- Microfuge
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- P10 pipette and tips
- P100 pipette and tips
- P1000 pipette and tips
- P2 pipette and tips
- P20 pipette and tips
- P200 pipette and tips
- Qubit RNA HS Assay Kit (ThermoFisher, cat # Q32852)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019)
- SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, cat # 18080044)
- T4 DNA Ligase 2M U/ml (NEB, cat # M0202M)
- Thermal cycler
- Timer
- Vortex mixer