Cdna synthesis system kit

Fruit samples were taken at six stages of fruit development from peach cultivar “Taijin Shui Mi”, including 20, 40, 60, 80, 100, and 120 days after bloom (DAB). Three biological replicates were collected for each stage. Fruit flesh was immediately frozen in liquid nitrogen and then ground to fine powder. Total RNA was extracted using a quick extraction kit (Aidlab, Beijing, China). First- and second-strand complementary DNA (cDNA) was synthesized using a cDNA Synthesis System kit (TOYOBO, Osaka, Japan), following the manufacturer’s protocol. Then double-strand cDNAs were purified and adapters were ligated to the short fragments. The constructed RNA-Seq libraries were sequenced using the Illumina HiSeq 2000 platform in paired-end 150-bp mode. Low-quality reads were filtered from the raw reads using Trimmomatic [73 (link)]. Data analysis followed the protocol proposed by Pertea et al. [74 (link)]. Cleaned reads were mapped to the peach reference genome using HISAT2 (Version 2.0.5) [75 (link)] using default parameters. Transcript abundances were estimated, and transcript assembly was performed using the Stringtie program [76 (link)]. DEG analysis was carried out using the R package ballgown [77 (link)].

Total RNA of 20 μg from each of the 18 RNA samples was sent to Novogene (Beijing, China) for construction of RNA-seq libraries and sequencing. The mRNA was purified from the total RNA using the Oligotex mRNA Midi Kit (Qiagen, Beijing), and assessed for quality using the Agilent Technologies 2100 Bioanalyzer (Agilent, United States). The mRNA was then broken into short fragment (approximately 300 bp). First and second strand complementary DNA (cDNA) was synthesized using a cDNA Synthesis System kit (TOYOBO, Japan) using random hexamer-primer, following the manufacturer’s protocol. Then double-strand cDNA were purified and adapters were ligated to the short fragments. The constructed RNA libraries were sequenced on the Illumina HiSeq^TM 2000 platform in paired-end (PE) mode.