RNA extraction, RNA-seq and quantitative real-time PCR analysis
Plant materials were collected
after 4 d co-cultivation. Frozen samples of the control and SQR9
VCs-treated seedlings were ground in liquid nitrogen and total RNA was
extracted with three biological replicates resulting in a total of 6
samples. Experimental protocols
for RNA sequence was performed according to the manufacture’s (Novogene
Experimental Department) technical instruction.
Differential expression analysis
of control and SQR9 VCs-treated samples was performed using the DESeq R
package (1.18.0). DESeq provide statistical routines for determining
differential expression in digital gene expression data using a model
based on the negative binomial distribution. The resulting
P-values were adjusted using the
Benjamini and Hochberg’s approach for controlling the false discovery
rate. Genes with an adjusted P-value < 0.05 found by DESeq
were assigned as differentially expressed.
The auxin-related differentially
expressed genes with a |log2(FoldChange)|
> 1 were selected for further analysis.
Three-day-old Arabidopsis seedlings of Col-0 grown on MS slats
agar medium were exposed to SQR9 VCs or not. After 4 d of
co-cultivation, total RNA was isolated from control or SQR9 VCs-treated
seedlings using Plant RNA Kit
R6827 (OMEGA bio-tek) following the manufacturer’s protocol. The
first-strand cDNA synthesis was generated from 1 μg of total RNA using
the HiScript 1st Strand cDNA Synthesis Kit (Vazyme).
Quantitative RT-PCR was performed
on a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using an
SYBR® Green Master Mix Kit (Vazyme).AtEF1A (AT5G60390) was used
as the reference gene and the primers are listed in Supplemental Table
S2. The results were obtained from three biological replicates and
2–ddCt was taken for every sample as the relative
expression levels. Values detected by quantitative RT-PCR are relative
to the lowest value for each gene.