*4.2. Methods of RNA Extraction and Detection*

The RNA samples were extracted with RNA Extraction Kit (RN40, Aidlab Biotechnologies, Beijing, China). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA), to ensure the use of qualified samples for sequencing.

Library preparation for sRNA sequencing: A total amount of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Ultra small RNA Sample Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. First of all, the 30SR Adaptor was ligated and mixed for Illumina. The RNA and nuclease-free water were mixed after incubation for 2 min at 70 ◦C in a preheated thermal cycler, which was then transferred to ice. A 30Ligation Reaction Buffer (2×) was then added and mixed with the 30Ligation Enzyme Mix, after which, the 30SR Adaptor was ligated and incubated for 1 h at 25 ◦C in a thermal cycler. To prevent adaptor–dimer formation, the SR RT Primer hybridizes to the excess of 30SR Adaptor (that remains free after the 30 ligation reaction) and transforms the single-stranded DNA adaptor into a double-stranded DNA molecule (dsDNAs) that is not a substrate for ligation. Subsequently, the 50SR Adaptor was ligated. Then, reverse transcription produced the synthetic first chain. Last, PCR amplification and Size Selection were performed. A polyacrylamide gel electrophoresis (PAGE) gel was used for fragment screening, rubber cutting recycling as the pieces get small RNA libraries. At last, the PCR products were purified (AMPure XP system, Beckman Coulter, Beverly, MA, USA), and the library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).

Library preparation for lncRNAs and circRNAs sequencing: A total amount of 1.5 µg RNA (for circRNA it was 2.0 µg) per sample was used as input material for rRNA removal using the Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA), following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, fragmentation was carried out using divalent cations under an elevated temperature in NEBNext First-Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using random hexamer primers and Reverse Transcriptase. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease and polymerase activities. After adenylation of the 3' ends of the DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. In order to select insert fragments of preferentially 150–200 bp (for circRNA it was 150–250 bp) in length, the library fragments were purified with AMPure XP Beads (Beckman Coulter). Then, 3 µL

USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 ◦C for 15 min before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index(X) Primer. At last, the PCR products were purified (AMPure XP system), and the library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR.
