title: RNA Seq of endophytic fungi in rice seeds
design: Environmental DNA of seed endophytic fungi was extracted from the prepared suspensions using the SteadyPure Plant Genomic DNA Extraction Kit (Accurate Biology Co., Ltd, Hunan, China). Fungal universal primers ITS1F (5'-CTT GGT CAT TTA GAG GAA GTA A-3') and ITS2R (5'-GCT GCG TTC TTC ATC GAT GC-3') were used for PCR amplification (Yang et al., 2024). Each 20 L PCR reaction contained: 10 L 2  Pro Taq mix, 0.8 L forward primer (5 M), 0.8 L reverse primer (5 M), and 10 ng template DNA. The PCR conditions were: 95 C for 3 min; 30 cycles of 95 C for 30 s, 58 C for 30 s, 72 C for 45 s; and a final extension at 72 C for 10 min. PCR products were purified using the PCR Clean-Up Kit (Axygen Co. Ltd, USA) and quantified with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Libraries were prepared using the NEXTFLEX Rapid DNA-Seq Kit (Shanghai Xinrui Biotechnology Co., Ltd, China) and sequenced on an Illumina NextSeq2000 platform.