The RPFs were 3 dephosphorylated with T4 PNK (New England Biolabs) for 1h at 37C to heal the 2-3 cyclic phosphate from the RNase I cleavage and were subsequently purified using the Oligo Clean and Concentrator kit (Zymo Research). The 3 adapter was ligated to the RPFs and the ligated fragments were purified following the protocol of the Small RNA Library prep kit (Lexogen). After the 5 phosphorylation of the RPFs and their purification with the Oligo Clean and Concentrator kit, 5 adapter ligation took place based on the Small RNA Library prep kit. First strand cDNA synthesis was performed according to the Small RNA Library prep kit manual. Ribosomal RNA (rRNA) was depleted with the addition of LNA probes from the QIAseq FastSelect rRNA HMR kit (Qiagen) during the cDNA synthesis reaction. Prior to Indexing PCR amplification, a qPCR was performed to determine the optimal PCR cycle number. During PCR, index primers (Lexogen) were added at both ends of the fragments to produce dual-indexed libraries. The PCR amplification protocol from the Small RNA Library prep kit was followed. Library products were purified with AMPure XP beads and their quality was determined on the Agilent Bioanalyzer with the DNA High Sensitivity kit (Agilent). For efficient removal of adapter dimers and excess index primers, the library products were run on a Novex TBE gel (8%) (Thermo Fisher Scientific) and the gel part corresponding to the library products was excised and purified. The quality of the final libraries was determined on the Agilent Bioanalyzer with the High Sensitivity DNA kit. The concentration of the libraries was estimated by performing qPCR using a dilution of PhiX Control v3 as standard and samples were pooled in an equimolar way. A test sequencing run (2 x 75 bp) on the Illumina MiSeq platform was performed with the MiSeq Reagent Nano kit v2 (Illumina) to check the quality of the prepared libraries prior to the in-depth sequencing on Novaseq