Rnase free dnase 1

The growth of ZS97 was maintained under routine field management in Wuhan (30°52′N, 114°32′E), China. To be consistent with the samples used in the previous microarray data (see Supplementary Table S1), the samples were harvested from ZS97 plants at the corresponding stages, including seedlings (three-leaf stage, Z12), young shoots (seedlings with two tillers, Z13), young roots (seedlings with 2 tillers, Z14), mature sheaths (secondary branch primordium differentiation stage, Z17), young flag leaves (5 days before heading, Z19), old flag leaves (14 days after heading, Z20), young panicles (4–5 cm, Z24), old panicles (heading stage, Z25), young stems (5 days before heading, Z26), spikelets (3 days after pollination, Z29), and endosperms (7 days after pollination, Z31). Calli (15 days after induction, Z4) were obtained after callus induction using ZS97 seeds. Total RNA was isolated from each sample with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. The total RNA was treated with RNase-free DNase I (New England Biolabs, Ipswich, MA, USA) to eliminate genomic DNA contamination. Quantitative real-time PCR (qRT-PCR) was performed with the same procedures as previously reported [11 (link)]. The primers used in the present study are listed in Supplementary Table S2.

Total RNA was extracted from larval and pupal samples by TRIzol (ThermoFisher Scientific, 15596026) following the manufacturer’s instruction. In brief, four groups of ten animals were grounded in 750 μl of TRIzol reagent and incubated at room temperature for 10 min. Phenol was removed from samples by multiple rounds of chloroform extraction. RNA from the supernatant was precipitated by adding 0.5× isopropanol and washed once with 70% ethanol. The extracted RNA was then treated with RNase-free DNase I (New England Biolabs, M0303) for 30 min at 37 °C to remove genomic DNA. Subsequently, DNase activity was heat-inactivated for 10 min at 65 °C upon adding 1 μl of 50 mM EDTA. The RNA was then reverse-transcribed with Oligo (dT)^{18 (link)} primer using a RevertAid First-strand cDNA synthesis kit (ThermoFisher Scientific, K1621). The relative expression level of genes was measured by qPCR and normalised to the expression level of housekeeping gene EF1α. For each qPCR reaction, 2× SensiFast SYBR Green PCR Master mix (Bioline, BIO-94005) was used in 20 μl reactions with 200 nM of each primer. The qPCR cycle was set as 95 °C for 10 min followed by 35 cycles of 95 °C for 30 s and 58 °C for 30 s. All primers used are listed in Supplementary Table 3.

Leaf samples were collected from 3 independent plants in each treatment of the 0, 1, 3, 6, 9, 12, 18, and 24 days post-inoculation (dpi), and they were immediately frozen in liquid nitrogen. Total RNA was isolated with Quick RNA isolation Kit (Huayueyang, Beijing, China) following the manufacturer’s recommended protocol. An aliquot of total RNA was treated with RNase-free DNase I (NEB, Ipswich, MA, USA) to degrade contaminating genomic DNA. The qualities and quantities of total RNA were assessed on NanoDrop ND-2000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The total RNA was subsequently reverse transcribed to double-stranded cDNA using Super Script III Reverse Transcriptase (TaKaRa, Dalian, China).

Total RNA extraction was achieved by the hot acid-phenol method. Briefly, cells were lysed and RNAs were extracted three times with an equal volume of acidic hot phenol and once with chloroform. After ethanol precipitation, RNAs were air dried and dissolved in water. Contaminating DNA was removed from total RNA by using 10 U of RNase-free DnaseI (New England BioLabs France) in a 50 μl mixture containing 6.25 mM MgCl₂ and approximately 3 μg μl^-1 of total RNA. The reaction mixture was incubated 30 min at 37°C, and the DNase I was then inactivated by adding 1 μl of 0.5 M EDTA to the mixture and incubating the mixture for 10 min at 65°C. The concentration was determined by measuring the absorbance at 260 nm. The quality of the RNA was then checked on a 2% agarose gel prior to use. Each RNA extraction was tested by PCR for the absence of contaminating DNA prior reverse transcription assays, and was performed in triplicate.