Next multiplex small rna library prep kit

NEB Next Multiplex Small RNA Library Prep Kit was utilized to prepare the libraries. First, 200 ng of total RNA was denatured under elevated temperatures. The denatured RNA was then ligated to a 3ʹ SR adapter, followed by SR-RT primer annealing to convert the free single-stranded adapter to the double-stranded DNA molecule. Reverse transcriptase was used to copy the 3ʹ SR adapter-ligated molecules into first-strand cDNA. Following that, the adapter-ligated products were purified and enriched using the thermal conditions provided here: initial denaturation at 94 °C for 30 s; 15 cycles at 94 °C for 15 s, 62 °C for 30 s, 70 °C for 15 s; final extension at 70 °C for 5 min. After that, PCR products were purified, and fragment size distribution was examined on Tape Station using D1000 DNA Screen Tapes (Agilent, Cat# 5067–5582), accompanied by size selection on 4% E-gels. The Qubit High Sensitivity Assay (Invitrogen, Cat# Q32852) was used to quantify the libraries that had been produced. Before cluster amplification on the Illumina flow cell, the resulting libraries were merged and reduced to the final optimum loading percentage. The cluster flow cell is then put into the HiSeq 2500 instrument to generate 25 M 50 bp single-end reads.

The nine replicate RNA samples (three from each stock) were used to construct nine small RNA libraries for small RNA sequencing, following the Next^® Multiplex Small RNA Library Prep Kit (NEB, Ipswich, MA, USA) manufacturer’s instructions. The nine sRNA libraries were sequenced on the Hiseq 2500 platform (2 × 75 bp read length, Illumina, San Diego, CA, USA) at Majorbio (Shanghai, China).