title: Illumina HiSeq 2000 sequencing; Identification of miRNAs-seq of 4-day broccoli sprouts and putative cross-kingdom regulated human target genes
design: Identification of miRNAs-seq of 4-day broccoli sprouts and putative cross-kingdom regulated human target genes
library_construction_protocol: Seeds of the sulforaphane-rich broccoli prototype Brassica rapa sylvestris (Megerle Online GmbH, Ubstadt-Weiher, Germany) were cultivated in a sterile beaker with distilled water at room temperature. After 4 days of germinating, the whole sprouts including the tiny roots were gently collected, minced with sterile scissors and rapidly frozen in liquid nitrogen.  performing PCR amplification according to manufacturer instructions NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible) (NEB, Cat No.: E7330). 94°C and 30 sec for initial denaturation，94°C and 15 sec for denaturation, 62°C and 30 sec for Annealing, 70°C and 15 sec for extension, 70°C and 5 min for final extension, followed by 4°C Hold Total RNA was extracted by Ambion® Plant RNA isolation aid, followed by isolation of miRNAs by the mirVanaTM miRNA isolation kit (both from Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer's instructions. The RNA concentration was evaluated with a NanoDrop 2000 Spectrophotometer.  Libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible) (NEB, Cat No.: E7330) following manufacturer instructions and using 100 ng total RNA obtained from 4-day broccoli sprouts.