2 Materials
2.1 Conversion of 5
0
Triphosphates to 5
0
Monophosphates
1. rRNA-depleted, high quality RNA (see Notes 2 and 3).
2. RNA 5
0 Pyrophosphohydrolase (RppH) and 10Â buffer (New
England Biolabs) (see Note 4).
3. RNase-free water.
4. RNA Clean & Concentrator-25 kit (Zymo Research)
(or alternative column-based RNA purification kit).
5. Murine RNase inhibitor (New England Biolabs) (or alternative
ribonuclease inhibitor).
6. 100 mM Tris pH 7.5, prepared with RNase-free water.
7. Vacuum microcentrifuge.
2.2 Adapter Ligation
1. Oligo SSS392: 5
0 TCC CTA CAC GAC GCT CTT CCG AUC
U 3
0 , where regular font indicates deoxyribonucleotides and
italic font indicates ribonucleotides, 5 μg/μL in RNase-free
water (see Note 5).
2. PEG 8000: 50% in RNase-free water.
3. Murine RNase inhibitor (New England Biolabs) (or alternative
ribonuclease inhibitor).
4. T4 RNA ligase I, standard concentration version
(10,000 units/mL) and 10Â buffer.
5. ATP: 10 mM in RNase-free water.
6. DMSO.
7. RNase-free water.
8. RNA Clean & Concentrator kit (Zymo Research)
(or alternative column-based RNA purification kit).
2.3 RNA
Fragmentation
and cDNA Synthesis
1. Oligo SSS397: 5
0 CTG GAG TTC AGA CGT GTG CTC TTC
CGA TCT NNN NNN 3
0 , where N indicates random deoxyribonucleotides, 1 μg/μL in RNase-free water.
2. NEBNext
® RNA First Strand Synthesis Module (includes Protoscript II reverse transcriptase, murine RNase inhibitor, and
reaction buffer containing dNTPs).
3. Thermocycler.
4. Tris pH 7.5: 1 M prepared with RNase-free water.
5. Actinomycin D: 0.1 mg/mL in DMSO.
6. NaOH: 1 M.
7. EDTA: 500 mM.
8. Tris pH 7.5: 1 M.
9. Monarch PCR Cleanup kit (New England Biolabs).
10. Molecular-grade water.
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