GSM1520981: P0_Root; Alhagi sparsifolia; RNA-Seq
Total RNA was extracted from P-0h, P-6h, P-24h and Rh-6h, respectively, by using TRIzol® reagent (Invitrogen, USA) Total RNA was first assessed with NanoPhotometer® spectrophotometer (IMPLEN, USA) and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA) to ensure the quality and integrity, and then processed with NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s instruction. Briefly, poly(A) mRNA was first isolated from 3 μg of total RNA using oligo (dT) magnetic beads (Invitrogen, USA), and then was fragmented into smaller pieces at 70℃ for 5min in the fragmentation buffer (Ambion, USA). Taking these mRNA fragments as templates, the first-strand cDNA was synthesized with M-MuLV Reverse Transcriptase (RNase H-) and random hexamer primer, and second strand cDNA synthesis was subsequently performed using DNA polymerase I (Invitrogen, USA) and RNaseH (Invitrogen, USA). The resulting double-stranded cDNA fragments were purified with AMPure XP system (Beckman Coulter, USA) followed by end repair, 3′ poly(A) addition, and NEBNext Adaptor ligation. The cDNA fragments of ~200 bp in length were selected and further enriched with PCR amplification to generate the cDNA libraries of P-0h, P-6h, P-24h and Rh-6h for Illumina sequencing. The library quality was evaluated on the Agilent Bioanalyzer 2100 system.