Second generation high throughput sequencing platform

Magnetic beads with oligo (dT) were used to enrich mRNA, and six-base random primers (random hexamers) were employed to synthesize cDNA. The double-stranded cDNAs were purified with AMPure XP beads, and PCR amplification was performed to construct the cDNA library. An Agilent 2,100 Bioanalyzer was used to detect the insert size of the library. After qualification, the Illumina second-generation high-throughput sequencing platform was used, and raw reads were filtered to obtain clean reads. Trinity (Grabherr et al., 2011 (link)) was used to assemble the transcripts of clean reads, and software RSEM (Dewey and Li, 2011 (link)) was used to calculate the gene expression using the FPKM method (Trapnell et al., 2010 (link)). Based on the average FPKM values under each treatment in roots and leaves, both the absolute values of the |log₂ (fold change)| ≥1 and the adjusted P-value < 0.05 were used as thresholds to identify DEGs.

RNA-Seq was performed by Allwegene Technology Co., Ltd. (Beijing, China). Total RNA was isolated from the samples of AH and AR using an RNeasy plant mini kit (QIAGEN, Inc., Valencia, CA, USA) and treated with DNase I (Roche, Inc., Branchburg, NJ, USA) to eliminate contaminating DNA, according to the instructions. The integrity of RNA was verified by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The mRNA content of samples was selectively enriched using magnetic Oligo (dT) beads, and a random hexamer was then used to target the mRNA as a template to synthesize the first-strand cDNA. Having obtained the first-strand cDNA, we proceeded to synthesize second-strand cDNA following the addition of DNA polymerase I, buffer, and dNTPs. AMPure XP beads were used to purify the resulting double-stranded cDNA, following the end repair and the addition of A-tails, and adapter sequences. Subsequently, double-stranded cDNA was size-selected via AMPure XP beads, and the cDNA library was constructed by PCR amplification. The sequencing of the cDNA to obtain raw read pairs was performed using the Illumina second-generation high-throughput sequencing platform based on the PE150 sequencing strategy.

After RNA quality control, RNA-seq was carried out with the HiSeq sequencing strategy using the Illumina second-generation high-throughput sequencing platform. Low-quality data were removed, and subsequently the filtered reads were aligned to the Mus musculus genome (GRCm38) using HISAT (Kim et al., 2015 (link)). The mapped reads were counted and converted to FPKM (Mortazavi et al., 2008 (link)) by RSEM (Li and Dewey, 2011 (link)).