manual NEBNext Multiplex Small RNA Library Prep Set for ... - URGV

Protocols:Libraries prepared by this method are compatible with paired-end flow cells.Starting Material: 100 ng–1 µg Total RNA.Ligate the 3´ SR AdaptorNote: For total RNA inputs of 100 ng, dilute the 3´ SR Adaptor for Illumina1:2 in nuclease free water.1. Mix the following components in a sterile nuclease-free PCR tube:Input RNA 1–6 µl3´ SR Adaptor for Illumina 1 µlNuclease-Free WatervariableTotal volume 7 µl2. Incubate in a preheated thermal cycler for 2 minutes at 70°C. Transfertube to ice.3. Add the following Components:3´ Ligation Reaction Buffer (2X) 10 µl3´ Ligation Enzyme Mix 3 µlTotal volume 20 µl4. Incubate for 1 hour at 25°C in a thermal cycler.Note: Longer incubation times and reduced temperatures (18 hours;16°C) increase ligation efficiency of methylated RNAs such as piwiinteractingRNAs (piRNAs) (if present in the sample). However, someconcatamerization products might be formed.Hybridize the Reverse Transcription PrimerThis step is important to prevent adaptor-dimer formation. The SR RTPrimer hybridizes to the excess of 3´ SR Adaptor (that remains free after the3´ ligation reaction) and transforms the single stranded DNA adaptor intoa double-stranded DNA molecule. dsDNAs are not substrates for ligationmediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptorin the subsequent ligation step.Note: For total RNA inputs of 100 ng, dilute the SR RT Primer for Illumina1:2 in nuclease free water.5. Add the following components to the ligation mixture from step 4 and mix well:Nuclease-Free Water 4.5 µlSR RT Primer for Illumina 1 µl4Total volume now should be 25.5 µl