Library preparation for small RNA sequencing (sRNA-Seq).

sRNA transcripts were converted into barcoded cDNA libraries. Library preparation was performed with a NEBNext multiplex small RNA library prep set for Illumina (protocol E7330; New England BioLabs Inc., USA) as described previously (22). For each sample, 250 ng of RNA was used as the starting material to prepare libraries. Each library was prepared with a unique indexed primer so that the libraries could all be pooled into one sequencing lane. Multiplex adapter ligations, reverse transcription primer hybridization, reverse transcription reactions, and the PCR amplification were performed as described in the protocol provided by the manufacturer. After PCR preamplification, the cDNA constructs were purified with a QIAQuick PCR purification kit (Qiagen, Germany) following the modifications suggested in the NEBNext multiplex small RNA library prep protocol. Further quality control checks and size selections were performed following the NEBNext multiplex small RNA library prep protocol (protocol E7330; New England BioLabs Inc., USA). Size selection of the amplified cDNA constructs was performed using Novex Tris-borate-EDTA (TBE) gels (Invitrogen) (6%) and following the procedure of gel electrophoresis running and purification of the construct described in the Illumina TruSeq small RNA library prep protocol. The 140-nt and 150-nt bands correspond to adapter-ligated constructs derived from RNA fragments of 21 to 30 nt. A concluding Bioanalyzer 2100 run performed with a high-sensitivity DNA kit (Agilent Technologies, Germany) permitted checking final size, purity, and concentration for the sequences in the DNA libraries.

The obtained libraries (24 samples were multiplexed) were subjected to the Illumina sequencing pipeline, passing through clonal cluster generation on a single-read flow cell (Illumina Inc., USA) by bridge amplification on a cBot (TruSeq SR cluster kit, v3-cBOT-HS; Illumina Inc., USA) and 50 cycles of sequencing by synthesis using a HiSeq 2000 sequencer (Illumina Inc., USA) (in collaboration with Genecore Facility at EMBL, Heidelberg, Germany).