Dp441 kit

Anthers of MT and WT at three different stage were collected with three biological replicates for RNA-seq. Total RNA was extracted from anthers using DP441 Kit (Tiangen, China) according to the manufacturer’s instructions. RNA integrity was assessed on a 1% agarose gel, and a Nanodrop 2000 spectrophotometer (Thermo, USA) was used for the measure of RNA concentration and contamination. Then, a total of 18 libraries of 3 stages in WT and MT were sequenced using Illumina HiSeqTM 2000 to generate paired-end reads. The reads files of this study are accessible from the NCBI Sequence Read Archive (SRA) database under Accession Number PRJNA598402.
Low quality reads were first removed, and the resulting clean reads were mapped to the upland cotton TM-1 genome using HISAT2 [41 (link)]. StringTie was used to calculate the fragments per kilobase of transcript per million mapped reads (FPKM) for estimating gene expression levels. Differential expression analysis was performed using the DESeq R package. Genes with FDR < 0.01 and normalized Fold Change≥2 were considered differentially expressed.

Floral buds were collected from the different growth stages. After removing the sepals, the remaining floral parts were immediately flash frozen in liquid nitrogen and stored at −80 °C. Three biological replicates were collected. Total RNA was extracted using the DP441 Kit (Tiangen, China). RNA quality was assessed on 1% agarose gels, and RNA purity was determined using a Nano Photometer spectrophotometer (IMPLEN, USA). RNA concentration was measured using a Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, USA). RNA-seq was performed by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China) on an Illumina HiSeq 4000 platform.
Raw reads were first filtered to obtain clean reads, which were aligned to the eggplant reference genome²⁵ using TopHat2. HTSeq (v0.6.1) was used to count the read numbers mapped to each gene. The FPKM of each gene was calculated. Differential expression analysis (three biological replicates per condition) was performed using the DESeq R package (v1.18.0). Genes with an adjusted P-value <0.05, as found by DESeq, were considered differentially expressed. GO enrichment analysis of DEGs was implemented using the GOseq R package. KOBAS software was used to test the statistical enrichment of DEGs in KEGG pathways.

Total RNA was extracted from each sample using a DP441 Kit (Tiangen, China), according to the manufacturer’s instructions. RNA quality was evaluated by 1% agarose gel electrophoresis, and RNA was purified using AMPure XP (IMPLEN, United States). RNA concentrations and RNA integrity were determined using a Qubit RNA Assay Kit and a Qubit 2.0 Fluorometer (Life Technologies, United States) and the RNA Nano 6000 Assay Kit with an Agilent Bioanalyzer 2100 (Agilent Technologies, United States), respectively. RNA was sequenced using the Illumina HiSeq high throughput sequencing platform (Illumina HiSeq 4000, United States). RNA extraction and transcriptome were completed by Beijing Novogene Bioinformatics Technology Co., Ltd., (Beijing, China).