2100 series

RNA libraries were prepared using the SureSelect Chain-specific RNA Library Preparation kit (Agilent Technologies). Thereafter, a fluorometer (Qubit 3.0; Thermo Fisher Scientific) and bioanalyzer (2100 series; Agilent Technologies) were used to quantify the purified libraries. Cluster generation was undertaken using cBot (Illumina, San Diego, CA, USA). Sequencing was achieved with NovaSeq™ 6000 (Illumina). These procedures were outsourced to Hefei Yuan En Biotechnology Co., LTD (Hefei, China).

Total RNA was extracted and purified by the mirVana™ miRNA Isolation Kit (catalog number: AM1561; Ambion, Austin, TX, USA) according to manufacturer instructions. RNA quality was evaluated by a spectrophotometer (Nanodrop™; Thermo Fisher Scientific, Waltham, MA, USA) and a bioanalyzer (2100 series; Agilent Technologies, Santa Clara, CA, USA).

TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) was employed to isolate and purify total RNA according to the manufacturer’s instructions. The amount and purity of total RNA in each sample was analyzed using a bioanalyzer (2100 series; Agilent Technologies, Santa Clara, CA, USA) and an RNA Nano1000 Assay Kit (Agilent Technologies) so that the RNA integrity number (RIN) > 7.0. Ribosomal RNA was removed according to the instructions of the Ribo-Zero™ rRNA Removal Kit (Illumina). At 95 °C, the remaining RNA was broken into short fragments using divalent cations. Subsequently, cDNA was created by reverse transcription using fragmented RNA as a template, followed by addition of deoxynucleoside triphosphate, RNase H, and Escherichia coli DNA polymerase I to synthesize the second strand. After purification of AMPure XP beads (Beckman Coulton, Fullerton, CA, USA) and end repair, poly(A) was added, and sequencing connectors were attached. Then, cDNA of a certain length range was extracted. PCR amplification was performed to obtain a cDNA library. The average insert size of the final cDNA library was 300 ± 50 bp. Twelve separate RNA-seq libraries were generated for the study group and control group.