GSM9238568: Rice whole roots 3 wpi, p35S, total mRNA, Myc(+Ri), repC; Oryza sativa; RNA-Seq
Promoter:TRAP constructs were prepared with the TRAP destination vector pH7WG-OsTRAP which was engineered by modifying p35S:HF-OsRPL18 (Zhao et al., 2017). This vector contains a Gateway recombination site for promoter insertion upstream of a chimeric ribosomal protein fusion comprising a 6×His-FLAG-3×Gly tag, GFP, and OsRPL18/eL18 (Os03G0341100). Promoters listed in Table S6 were cloned into pENTR-D/TOPO (Invitrogen) and subsequently recombined into the TRAP vector via LR Clonase II enzyme mix (Invitrogen). All constructs were validated by Sanger sequencing. Resulting T-DNA plasmids were introduced into Oryza sativa japonica cv. Nipponbare embryogenic calli, derived from mature seed embryos, through Agrobacterium tumefaciens-mediated transformation (strain EHA105A). T2 generation plants were used for TRAP experiments. TRAP was performed as previously described (Mustroph et al., 2009; Reynoso et al., 2015) with modifications outlined by Reynoso et al. (2019). From each sample, both total RNA and TRAP RNA (following ribosome purification) were extracted using the RNeasy Micro Kit (Qiagen). Libraries were prepared with the QuantSeq 3' mRNA-seq kit (Lexogen) incorporating unique dual indices (UDIs) and unique molecular identifiers (UMIs) for three biological replicates per treatment. RNA samples were diluted to an equal concentration and sent for poliA library generation and sequencing to Novogene (Cambridge) with a sequencing depth of 3 Gb (10 million reads). Sequencing was performed on an Illumina NovaSeq 6000 platform generating 150 bp single-end reads.