Dntp mix

Single-stranded cDNA was obtained through the same procedures as described in the SLiPiR-seq library preparation section. qPCR was performed based on the S-Poly(T) Plus method: 1 μl of cDNA template (1:25, 1:10, 1:10, 1:20, 1:2, 1:20, 1:2, 1:20, and 1:10 dilution of original cDNA for lncRNA, miRNA, piRNA, mRNA, snRNA, rsRNA, snoRNA, ysRNA, and tsRNA, respectively), 0.2 μl of AceTaq DNA Polymerase (Vazyme), 2 μl of 10× AceTaq Buffer (Vazyme), 0.4 μl of 10 mM dNTP Mix (Vazyme), 0.2 μl of 100× ROX (Sigma-Aldrich), 0.5 μl of 5× SYBR Green (Roche Diagnostics), 5 μl of 1 μM universal reverse primer (5’-TACGAGATGTGACTGGAGTT-3’), 5 μl of small RNA specific forward primer (Supplementary Data 1), and add double-distilled water up to 20 μl. Amplification conditions were set as follows: activation at 95 °C for 5 min, followed by forty cycles of 95 °C for 10 s, 60 °C for 30 s. Assays were performed on ABI StepOne plus a real-time PCR system (Applied Biosystems).

The one-step RT-qPCR assay (referred to as our RT-qPCR assay) was performed in 96-well plates with a reaction volume of 30 μL. For the assay, 3 μL of 10× Taq Buffer, 0.24 μL of Champagne Taq DNA Polymerase, 0.35 μL of HiScript II Reverse Transcriptase, 0.25 μL of Murine RNase inhibitor, 0.3 μL of heat-labile UDG, 2.4 μL of MgCl2 and 0.6 μL of dNTP mix (Vazyme Biotech Co., Ltd., Nanjing, China), 0.6 μL of forward primers and 0.6 μL of reverse primers for each gene, 0.15 μL of probes for each gene, 13.81 μL of nuclease-free water, and 5 μL of template were mixed per reaction. The cycling conditions for each assay consisted of incubating the template at 55 °C for 5 min and 95 °C for 30 s, followed by 45 cycles of RT-qPCR, with each cycle consisting of 95 °C for 5 s and then 62 °C for 20 s. All reactions were carried out with a SLAN^®-96S Real-Time PCR System (Hongshi Medical Technology Co., Ltd., Shanghai, China).