2.3 Samples for RNA sequencing and bioinformatics analysis

Samples for RNA extraction and sequencing were collected at true leaf-age four: the third leaf and tissues near shoot apical meristem (SAM, after removal of outside 2 layers of sheath keeping about ±3 mm of the growth point) were collected swiftly on ice and snap frozen in liquid N2, then stored at -80°C till use. Tillers are supposed to start to appear from the leaf axillary zone at this stage, therefore, the SAM were collected for the RNA sequencing to reflect tiller-related genes profile. RNA extraction, pre-treatment, sequencing library generation and bioinformatics algorithm all followed protocols of a previous report (Zhang et al, 2019). We chose four treatments from two [CO2] combined with two N application rates (N0 and N10) for RNA sequencing analysis. Three independent biological replication samples for each treatment was sequenced, and the acquired RNA sequencing data were no less than 6 Gb bases per sample, which were qualified for gene expression analysis (Table S1).
The filtered data were used in the mapping and expression analysis following previous protocol (Trapnell et al, 2012). The FPKM (Fragments Per Kilo base transcript per Million mapped reads) value was used to indicate the expression level of a gene, which was calculated by this equation: total exon reads / (mapped reads (millions) * exon length (kilo base)). Differentially expressed gene (DEG) was defined as genes showing the ratio of average FPKM between two groups surpassed two-fold (≥2, or ≤0.5) and the adjusted false discovery rate (adj FDR) beingp ≤0.05.