title: RNA-seq of rice: Root under drought stress
design: 1. Experimental Procedure 1.1 Sample quality control Please refer to QC report for methods of sample quality control. 1.2 Library preparation for Transcriptome sequencing Non strand specific library Messenger RNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the secondstrand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Thelibrary was checked with Qubit and real-time PCR for quantificationandbioanalyzer for size distribution detection. Strand specific library Messenger RNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strandcDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size 1.3 Clustering and sequencing 16 After library quality control, different libraries were pooled basedontheeffective concentration and targeted data amount, then subjectedtoIllumina sequencing. The basic principle of sequencing is "SequencingbySynthesis", where fluorescently labeled dNTPs, DNA polymerase, andadapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends its complementary strand, theaddition of each fluorescently labeled dNTP releases a correspondingfluorescence signal. The sequencer captures these fluorescence signals and converts them into sequencing peaks through computer software, thereby obtaining the sequence information of the target fragment.  selection, USER enzyme digestion, amplification, and purification. Thelibrary was checked with Qubit and real-time PCR for quantificationandbioanalyzer for size distribution detection.