- 0.2 mL RNase-free PCR tubes, strips of 8.
- Flat PCR Caps, strips of 8.
- PCR thermal cycler.
2.8 RNA Purification 1. RNeasy MinElute Cleanup kit (Qiagen).
- 96% ethanol.
- 80% ethanol.
2.9 Library
Preparation Using
NEBNext®Multiplex
Small RNA Library
Prep Set for Illumina®
- NEBNext® Multiplex Small RNA Library Prep Set for
Illumina®(set 1 or 2, New England Biolabs) (seeNote 2). - 0.2 mL PCR tubes, strips of 8.
- Flat PCR Caps, strips of 8.
- PCR thermal cycler.
2.10 Library
Purification Using
GeneJET PCR
Purification Kit
- GeneJET®PCR Purification kit or equivalent.
- RNase-free 1.5 mL microcentrifuge tubes.
- Tabletop centrifuge.
- 1.5 mL DNA low-binding tubes.
2.11 Library
Quantification and
Quality Assessment
2.11.1 Library
Quantification
- Fluorometer able to quantify DNA library with high sensitivity
(e.g., Qubit®2.0 fluorometer). - Qubit®dsDNA HS Assay kit (0.2–100 ng).
- Thin-walled polypropylene tubes of 500μL compatible with
the fluorometer (e.g., Qubit®Assay Tube or Axygen®PCR-
05-C tubes).
2.11.2 Library Quality
Assessment
- Agilent 2100 Bioanalyzer (Agilent Technologies).
- Agilent HS DNA kit (quantitative range 5–500 pg/μL).
- Chip priming station (Agilent Technologies).
- RNase-free 1.5 mL microcentrifuge tubes.
2.12 Library
Sequencing
- An Illumina sequencer (starting from MiSeq to different HiSeq
models). - Any appropriate sequencing kit for a single read length of
35–50 nt.
3 Methods
3.1 Yeast rDNA PCR
Amplification
- Mix in a PCR tube 1μL of pHW18 template, 0.5μL of each
corresponding forward and reverse primers, 5μL Pfu DNA
polymerase buffer, 8μL dNTP mix, and 1μL Pfu DNA poly-
merase in a total volume of 50μL.
34 Lilia Ayadi et al.