Rna extraction kit

RIL^pur–W22 and RIL^bro–W22 seedlings (including shoots and roots, with the seeds removed) germinated for 14 days in 0 mM and 100 mM NaCl solution environments for RNA sequencing, with two replicates for each sample. Total RNA was extracted using the RNA extraction kit (Mei5bio, Beijing, China). RNA concentration and quality were measured using an ultra–micro spectrophotometer ND2000 (Thermo Scientific, New York, NY, USA). The prepared RNA was sent to Annoroad Gene Technology (Beijing, China) for library construction, sequencing, and data filtering (Table S9). Using the DNBSEQ–T7 sequencing platform, and the libraries were sequenced with a read length of 150 bp (pair–end).

Approximately 30 seeds (20 DAP) of purple (WT) and bronze (bronze mutant) color were isolated from an F₂ segregated population. Pericarp removed mutant and WT seeds were pooled together respectively and subsequently ground in liquid nitrogen. Total RNA of two pools was extracted with an RNA extraction kit (Mei5bio, Beijing, China). followed by the QC (quality control) with Agilent2100. Then, we performed pair end sequencing on an Illumina HiSeq4000 platform. Raw sequencing data and clean data with trimmed barcodes and primers were obtained from Annoroad (Beijing, China). Single reads that uniquely mapped to the genome were retained for single nucleotide polymorphism (SNP) calling. Identification of SNPs linked to the target gene was performed following the parameters described by published method [56 (link)].