title: GSM3208224: H4ck2; Boehmeria nivea; RNA-Seq
library_construction_protocol: To prepare samples for Illumina sequencing, total RNA of each sample was extracted using an EASYspin plus Total RNA kit (Aidlab, Beijing, China) following the protocol. Quality and yield of RNA were determined by agarose gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), respectively. Total RNA was extracted from the stem samples with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified with Plant RNA Purification regent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The concentration and purity of total RNA was determined using NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was examined by Agilent2100 bioanalyzer (Agilent, Santa Clara, CA, USA).Illumina sequencing was performed at Majorbio Bio-pharm Technology Co. Ltd, Shanghai, China (http://www.majorbio.com/). For transcriptome sequencing, eight individual libraries (Yy97ck1, Yy97ck2, Yy97tm1, Yy97tm2, Hdck1, Hdck2, Hdtm1 and Hdtm2) were constructed using 5 μg of total RNA. Briefly, poly (A) mRNA was extracted from the total RNA sample using Oligo (dT) magnetic beads. mRNA was fragmented into 200–500 nt pieces by adding a fragmentation buffer. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Life Technologies, Inc.) and random hexamer primers. After generation of second-strand cDNA, the double-strand cDNA was end-repaired, and a single 'A' base and indexed adapters were ligated to the fragments. The cDNA library was constructed with the Illumina Paired End Sample Prep kit (Illumina, USA), quantified by TBS380 (Picogreen, Invitrogen, USA) and was then sequenced on the Illumina HiSeqTM2500 (2×150 bp read length) platform.