2.3. miRNA library preparation

Libraries of small RNAs from each sample were prepared from total RNA using the NEBNext Small RNA Library Prep Set for Illumina (New England BioLabs Inc.) according to manufacturer's protocol. Briefly, 3′ adapters were ligated to total input RNA followed by hybridization of multiplex SR RT primers and ligation of multiplex 5′ SR adapters. Reverse transcription (RT) was performed using ProtoScript II RT for 1 h at 50 °C. Immediately after completion of the RT reaction, PCR amplification was performed for 15 cycles using LongAmp Taq 2X master mix. Illumina indexed primers were added to uniquely barcode each sample. Post- PCR material was purified using the QIAquick PCR purification kit (Qiagen). Post-PCR yield and concentration of the prepared libraries were assessed using a Qubit 2.0 Fluorometer (Invitrogen) and DNA 1000 chip on an Agilent 2100 Bioanalyzer (Applied Biosystems), respectively. Size selection of small RNA was done using 3% dye free agarose gel cassettes on a Pippin prep instrument (Sage Science Inc., Beverly, MA, USA). Post-size selection concentration and size estimation of the libraries were assessed using Qubit 2.0 Fluorometer and DNA High sensitivity chip on Agilent 2100 Bioanalyzer, respectively. Accurate quantification for sequencing applications was performed using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems). Each library was diluted to a final concentration of 1.25 nM and pooled in equimolar ratios prior to clustering.