RNA Extraction and Sequencing Protocol

Total RNA was extracted using TRIzol reagent (Thermo Fisher, 15,596,018) as previously reported [12 (link)]. Total RNA quantity and purity were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067–1511). Additionally, mRNA was purified from total RNA (5 μg) using Dynabeads Oligo (dT) (Thermo Fisher Scientific, CA, USA) with two rounds of purification and fragmented into short fragments using divalent cations at an elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA) at 94 °C for 5–7 min). Then, the cleaved RNA fragments were reverse-transcribed to create cDNA with SuperScript™ II Reverse Transcriptase (Invitrogen, cat.1896649, USA). Dual-index adapters were used and size selection was performed by using AMPureXP beads. The U-labeled second-stranded DNAs were treated with the heat-labile UDG enzyme (NEB, cat.m0280, USA). PCR was used to amplify to the ligated products under the following conditions: 95 °C for 3 min; 8 cycles of denaturation at 98 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s; and 72 °C for 5 min. Finally, 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China).