Oligo dt beads

Total RNA from primary cultured neurons after 1,3,5,7,10 days of infection was extracted using Multisource Total RNA Miniprep Kit (Axygen, 365). RNA quality was performed using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and checked using RNase free agarose gel electrophoresis. After qualification, total mRNA was enriched by Oligo (dT) beads (Epicentre, USA). RNA was fragmented into short fragments and reverse transcript into cDNA with random primers. Second-strand cDNA were synthesized and the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Netherlands), end repaired, poly A added, and ligated to Illumina sequencing adapters. Selecting ligation products with proper size by PCR and agarose gel electrophoresis, and then sequencing was performed by Gene Denovo Biotechnology Co. (Guangzhou, China) on Illumina HiSeq2500.

The RNA sequencing assay was performed by Gene Denovo Biotechnology (Guangzhou, China). Total mRNAs were extracted from each cell line using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The RNA quality was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and RNase free agarose gel electrophoresis. The total mRNAs were further enriched by Oligo(dT) beads (Epicentre, Madison, WI, USA) and fragmented into short fragments (about 200 nucleotides) using fragmentation buffer. Subsequently, the fragmented mRNAs were reverse-transcribed into cDNA using random primers, DNA polymerase I, RNase H, dNTP and buffer. After purification, repairing, ligating the cDNA with the Illumina sequencing adapters, agarose gel electrophoresis and PCR assay, the products were sequenced using Illumina Novaseq 6000 (Gene Denovo Biotechnology, Guangzhou, China).

Construction of mRNA library and sequencing were performed as described previously [15 (link)]. Briefly, total mRNA was enriched using Oligo (dT) beads (Epicenter, Madison, WI, USA), fragmented, and reverse-transcribed. Second strand cDNA was obtained using DNA polymerase I, RNase H. Then the cDNA fragments were purified using a QIAQuick PCR Extraction Kit (Qiagen, Hilden, Germany) and end-repaired; a poly(A) tail added and then ligated to Illumina sequencing adapters. The ligation products were finally sequenced using Illumina HiSeq TM2500 by Gene Denovo Biotechnology Co (Guangzhou, China).

For ‘Sijigui’, buds at S0 and S1 stages in winter were collected on November 15 and December 1, respectively (referring to the third flowering process in Figure 2), and the S0 and S1 buds in early summer were collected on June 1 and June 15, respectively (referring to the first flowering process in Figure 2). For ‘Yanhonggui’, buds at S0 and S1 stages in early summer were also collected on June 1 and June 15, respectively (referring to the flowering process in Figure 1A). RNA from the six tissues was extracted. Prokaryotic mRNA was enriched by eliminating rRNA using the Ribo-Zero™ Magnetic Kit, whereas eukaryotic mRNA was enriched with Oligo(dT) beads (Epicentre). With the use of random primers and fragmentation buffer, the enriched mRNA was reverse transcribed into cDNA. DNA polymerase I, RNase H, dNTP, and buffer were used to create second-strand cDNA. The cDNA fragments were then end repaired and poly(A) added. Illumina sequencing adapters were ligated after they had been purified using a QIAquick PCR extraction kit. Gene Denovo Biotechnology Co. used agarose gel electrophoresis to size-select the ligation products before PCR amplification and Illumina HiSeq™ 4000 sequencing (Guangzhou, China).

Total RNA from the lungs was extracted using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, and eukaryotic mRNA was enriched by oligo (dT) beads (Epicenter, Madison, WI, USA). RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified by 1% gel electrophoresis. All samples presented an RNA integrity number (RIN) > 7.5.