BGISEQ-500 paired end sequencing; Rice (Oryza sativa ssp. japonica variety KitaakeX) leaf transcriptome analysis transgenic lines overexpressing dTALEs
Rice (Oryza sativa ssp. japonica variety KitaakeX) leaf transcriptome analysis transgenic lines overexpressing dTALEs
To minimize biological variation due to sampling error, three biological replicates of each ZjPCKpro:dTALE1-STAP:GUS transgenic event were grown. T2 seed derived from three homozygous T1 lines for each independent insertion event were used – i.e. STAP4 - lines 4.2, 4.5, 4.6;  STAP45 - lines 17.3, 17.5, 17.9; STAP56 - lines 21.3, 21.5, 21.8; and STAP62 - lines 23.1, 23.3, 23.8 & lines 24.3, 24.6, 24.8. Seed were sterilized and germinated on MS-hyg media (2.15 g/L MS salts & vitamins, 15 g/L sucrose, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES) pH 5.8,  2 g/L phytagel, 20 mg/L hygromycin) in a growth cabinet with a 16 h light/8 h dark photoperiod, light intensity of 150 μmol photons m−2 s−1, 24oC constant temperature and relative humidity of 60%. After 7 days, plants were transferred into 0.5 L pots with soil and grown in the controlled environment chamber until the 4th leaf was fully expanded. Leaf tissue (0.5 cm2) from the mid-distal leaf blade portion of the 4th fully expanded leaf was collected from ten plants per line, pooled into a 2 mL centrifuge tube, frozen in liquid N2 and stored at minus 80oC. For each line, five technical replicate pools were obtained. Samples were collected between 9am and 11am on the same day. Frozen samples were homogenized using a TissueLyser II.  For dTALE2-STAP2:GUS lines, T1 and wild-type seeds were sterilized and germinated in soil at 28°C with 16 h light (400 μmol photons m−2 s−1) and 8 h dark cycle. Two independent T1 lines each of ZjPCKpro:dTALE2-STAP2.4:GUS and ZmPEPCpro:dTALE2-STAP2.4:GUS were grown, plus one T1 line of ZmPEPCpro:mTurquoise-STAP2.4:GUS. DNA was extracted from leaf 2 of 21 day old seedlings for genotyping by PCR, using  HptF: CGCAAGGAATCGGTCAATACA and HptR: GATGCCTCCGCTCGAAGTAG primers to amplify the hpt transgene sequence. Plants with confirmed transgene insertions were selected for RNAseq analysis. For each T1 line, leaf 4 was collected from three individual 21 day old seedlings. For the dTALE1 lines RNA was extracted using an RNeasy Plant Mini Kit. DNA was removed from the samples using an Ambion TURBO DNA free kit and RNA quality was determined using a NanoDrop.   For the dTALE2 lines RNA was extracted using Trizol reagent (Invitrogen, Waltham, Massachusetts, US) and the RNeasy Plant Mini Kit. The quality of total RNA was checked using an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, US) by the Institute of Molecular Biology Genomics Core at Academia Sinica.  library construction was performed by BGI Tech Solutions Co., Limited (Tai Po, N.T., Hongkong)