Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 229

with 0.6 m Lof3 0 -RNA adaptor (Illumina part #1000596), 1 m Lof10 T4 RNA
ligase buffer, 1 m L of RNaseOUT, and 1 m L of T4 RNA ligase. Incubate the reaction
mixture at 20 C for 6 h in a thermal cycler and hold overnight at 4 C. Separate
ligated RNAs by electrophoresis on a 15% TBE-urea gel along with 20-100 bases
ladder and recover RNAs corresponding to 70-90 nt bands using the same elution
and ethanol precipitation steps mentioned above. Dissolve the RNA pellet in ultra-
pure water.
2. Adaptor Ligation (Alternative)
Dissolve purified small RNA in 5 m L of ultrapure water and mix with 1 m Lof1
preadenylated 3 0 -sRNA adaptor (Illumina part #1000263). Incubate at 70 C for
2 min then immediately transfer onto ice. Add 1 m Lof10 T4 RNL2 truncated
reaction buffer (NEB), 0.8 m L of 100 mM MgCl 2 , 1.5 m L of T4 RNA ligase 2
truncated (NEB), and 0.5 m L of RNaseOUT. Incubate the reaction mixture in a
thermal cycler at 22 C for 1 h. Then add 1 m Lof5 0 -RNA adaptor (Illumina part
#1000595), 1 m L of 10 mM ATP, and 1 m L of T4 RNA ligase. Incubate the reaction
mixture in a thermal cycler at 20 C for 1 h. Store the product at 4 C.
3. Reverse Transcription and PCR Amplification
Mix 4.5 m L of adaptor-ligated RNAs and 0.5 m L of RT primer (Illumina part
#100597). Heat the mixture in a thermal cycler at 65 C for 10 min and transfer
immediately onto ice. Add 2 m Lof5 first strand buffer (Invitrogen), 0.5 m Lof
12.5 mM dNTP mix, 1 m L of 100 mM DTT and 0.5 m L of RNaseOUT. Heat the
sample to 48 C in a thermal cycler for 3 min. Add 1 m L of SuperScript II Reverse
Transcriptase (Invitrogen) and incubate the reaction mixture in a thermal cycler at
44 C for 1 h. The final volume is 10 m L. Premix 40 m L of PCR master mix with
28 m L of ultrapurewater, 8 m Lof5 Phusion HF buffer (Finnzymes), 0.5 m LofPrimer
GX1 (Illumina part # 1000591), 0.5 m L of Primer GX2 (Illumina part #1000592),
0.5 m Lof25mMdNTPmix,and0.5 m L of Phusion DNA Polymerase (Finnzymes).
Mix 10 m L of reverse-transcribed cDNAs and 40 m L of PCR master mix and perform
the PCR reaction using the following protocol: (1) 30 s at 98 C,(2)15cyclesof10sat
98 C, 30 s at 60 C, and 15 s of 72 C, (3) 10 min at 72 C, (4) hold at 4 C. Purify the
PCR products (a band of approximately 92 bp in length) by separating on a 6% TBE-
urea gel followed by elution and ethanol precipitation. Resuspend purified cDNA
libraries in 10 m L of resuspension buffer (Illumina part # 1001388). Use 1 m Lofthe
product for TOPO cloning and validate 100 colonies with traditional sequencing.
J. Hybridization, Cluster Generation, and Sequencing
The following approaches require operators specifically trained in the use of
Illumina Genome Analyzer. An average-size C. elegans laboratory may seek assis-
tance or use the service of a core facility. Briefly, denature the cDNA templates by