Illumina HiSeq 2000 paired end sequencing; RNA seq of early flowering phenotype se13 mutant, G123, at 20h after dawn
RNA isolation for RNA-seq analysis was performed using the NucleSpin RNA plant kit (Ref: 740949.50, MACHEREY-NAGEL, Germany) following the manufacturer's instructions. The quality and concentration of the RNA were tested by agarose gel electrophoresis and by using BioAnalyzer 2100 (Agilent) and NanoDrop spectrophotometer (Thermo Scientific). mRNA was enriched using oligo-dT beads. After random mRNA fragmentation, cDNA was synthesized by using random hexamer primers and reverse transcriptase, followed by the synthesis of the complementary strand following the Illumina mRNA Sequencing Sample Preparation Guide. Then, a series of end terminal repair and ligation of the pair-end sequencing adaptors were performed. The adapters used were: 5' adapter 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT and 3' adapter 5'- GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(ATCACG)ATCTCGTATGCCGTCTTCTGCTTG, where the 6 bases wrapped in parentheses corresponded to the index. Finally, size selection and enrichment by PCR amplification were performed. The quality testing of the library was tested by using Qubit 2.0 and Bioanalyzer 2100 and the effective concentration of the library was quantified accurately by Q-PCR.