GSM5321804: Hyp.pif457.LRFR.02; Arabidopsis thaliana; RNA-Seq
For RNA isolation, 5d-old seedlings were harvested in liquid nitrogen and kept in -70 °C for overnight. Next day, seedlings were covered with -70 °C cold RNAlater™-ICE (Thermo Fisher Scientific, United States, AM7030) and transferred to -20 °C for overnight. Cotyledons and hypocotyls are dissected using sharp needles on top of an ice block under a binocular microscope (Nikon, SMZ1500) and RNA isolation is done using RNeasy Mini Kit (Qiagen, Cat. No. / ID: 74004). For the RNA-sequencing, RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). From 40 ng total RNA, mRNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq libraries were then prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Massachusetts, USA). Libraries were quantified by a fluorimetric method and their quality assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with the resulting libraries using Illumina HiSeq 3000/4000 SR Cluster Kit reagents. Libraries were sequenced on the Illumina HiSeq 4000 with HiSeq 3000/4000 SBS Kit reagents for 150 cycles. Sequencing data were demultiplexed with the bcl2fastq Conversion Software (v. 2.20, Illumina; San Diego, California, USA).