NEBNext® Multiplex Small RNA Library Prep Set for Illumina®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Starting Material: 100 ng–1 µg Total RNA. Small RNA fragments should have a 5´ phosphate and 3´ OH to ligate and must be free of ATP.

Protocol tips
Starting Material: 100 ng–1 µg Total RNA. Small RNA fragments should have a 5´ phosphate and 3´ OH to ligate and must be free of ATP.

Publication protocol

Total RNA extractions were performed according to manufacturer’s instructions using TRIzol and TRIzol LS (both Thermo Fisher Scientific, Waltham, MA, USA) for cells and EVs respectively. Improved RNA precipitation was gained by adding 2 µl of PolyAcryl Carrier PC 152 polymer (Molecular Research Center, Inc., Cincinnati, OH, USA) per reaction. The RNA integrity of the cell samples was verified on Bioanalyzer RNA 6000 Pico Total RNA Kit (both Agilent Technologies, Lanarkshire, UK) and the RNA concentration for all samples was measured with the Qubit 2.0 Flurometer by using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). 250 ng of total RNA was subjected to small RNA library preparation by using the NEBNext Multiplex Small RNA Library Prep Set 1 for Illumina (NEB, Ipswich, MA, USA) kit according to the manufacturer’s instructions. The barcoded samples were size selected on the 6% Novex TBE PAGE gel (Thermo Fisher Scientific, Waltham, MA, USA) and the fragments corresponding to microRNA range were cut and subjected to purification with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). Thereafter, the products were quantified by using the KAPA Library Quantification Kit (Cat.No.KK4824, Kapa Biosystems, London, UK) and pooled at equimolar ratio. Two libraries (technical replicates) were generated in parallel, each one eventually containing a pool of 12 barcoded samples. The readymade libraries were further checked on the High Sensitivity D1000 ScreenTape (Agilent Technologies, Lanarkshire, UK) and quantified by using the KAPA Library Quantification Kit to enable precise loading of the flow cell. The clusters were generated by using the cBot and one replicate per lane was sequenced on the HiSeq 2500 (HiSeq Control Software 2.0.12.0/RTA 1.17.21.3, Illumina Inc., San Diego, CA, USA) with a 1 × 51 setup in RapidRun mode.

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