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Small RNA-seq library preparation and sequencing

MS Marta Seco-Cervera
DG Dayme González-Rodríguez
JI José Santiago Ibáñez-Cabellos
LP Lorena Peiró-Chova
FP Federico V Pallardó
JG José Luis García-Giménez
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Small RNA samples were converted to Illumina sequencing libraries using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1&2) (New England Biolabs, MA, USA), following the manufacturer’s protocol. Briefly, 5′ and 3′ adapters were ligated with small RNA molecules purified from plasma, followed by a cDNA library construction and incorporation of index tags by reverse transcription-PCR (RT-PCR). The products of this RT-PCR were purified using 6% non-denaturing polyacrylamide gel electrophoresis, and then size selection of 145–160 bp fraction was performed. The cDNA library samples were hybridized to a paired end flow cell and individual fragments were clonally amplified by bridge amplification on the Illumina cBot cluster generation. Then, the flow cell was loaded on the HiScanSQ platform and sequenced using Illumina’s sequencing by synthesis chemistry, generating 50 bp single end reads.

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