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.