title: RNA-Seq of soybean: seed coat
design: RNA was extracted via the TRIzol method, and RNA integrity was detected via 1% agarose gel electrophoresis. The RNA was stored in a -80 C freezer, after which the extracted total RNA was transferred to Beijing Histology Biotechnology Co., Ltd. (Beijing, China) for sequencing on dry ice. The extracted RNA was fragmented via a PCR plate with a magnetic plate holder. Fragmented mRNA was reverse transcribed into cDNA via Superscript II and random primers (Invitrogen, Carlsbad, CA, USA). The RNA-seq library preparations were sequenced on an Illumina HiSeq 2500 platform, and 150 bp paired-end reads were generated. Data filtering and quality control were performed through fastp software, and the resulting clean data were used for subsequent analysis. The soybean genome (http://plants.ensembl.org/Glycine_max/Info/Index) was used as a reference, and TopHat2 was used for read comparison. The Cuffquant and Cuffnorm components of Cufflinks software were used to quantify the expression levels of transcripts through the position information of mapped reads on genes. Gene expression was measured via the fragments per kilobase of transcript per million mapped reads (FPKM) method.