sRNA_DAF15-1 (CRX1174168)
Total RNA was isolated and purified following the manufacturer's procedure. The RNA amount and purity were evaluated using the NanoDrop ND-1000 (NanoDrop, Wilmington, DE). The integrity of RNA fragments was assessed using the Bioanalyzer 2100 (Agilent, CA). The obtained total RNA was denatured at 70C for 2 minutes with DNA 3' adapters, and then mixed with T4 RNA Ligase 2, truncated with K227Q (NEB, M0351L) at room temperature. The 3' adapter ligation reaction was performed at 16C for more than 8 hours. The 5' adapters and the products from the previous step were ligated with T4 RNA Ligase (NEB, M0204L) at 37C for 60 minutes. The reverse-transcription reaction was performed using SuperScript II Reverse Transcriptase (Thermo Fisher, 18064014). The cDNA products were amplified using Phusion High-Fidelity DNA Polymerase (NEB, M0530L). The PCR products were purified and enriched through PAGE electrophoresis. The sequencing strategy was single-end 50 bp on an Illumina HiSeq 2500 (LC-Bio Technology CO., Ltd., Hangzhou, China), following the vendor's recommended protocol.