Total RNA was isolated with TRIzol (Invitrogen) from each sample according to the manufacturer’s instructions and stored at -80℃. At the beginning of all below experiments, total RNA was treated for 30 minutes at 37℃ with RNase-free DNase I (New England Biolabs, NEB) to remove residual DNA.
Description
We chose RNA from panicle at filling stage to construct mRNA tag library, 6μg total RNA was consumed. Firstly, mRNA (PolyA+ RNA) was enriched by the Dynalbeads™ Oligo(dT)25 (Invitrogen) according to the manufacturer’s instructions. At the last step, beads with mRNA were suspended in 1×first strand buffer. Instead of elute mRNA from magnetic beads; the process of reverse transcription was performed on the beads, and the oligodT linked to beads was used as primer. We introduced the following reagent ordinal for first strand cDNA synthesis: 0.1M DTT, and 10mM dNTPs (NEB), 40U of RNaseOUT™, 400U of Superscript II reverse transcriptase (Invitrogen), and added DEPC H2O to a final volume of 50 μl. This reaction mix was incubated at 42°C in a thermomixer spin at 1400rpm for 1 hours sustainedly, followed by inactivate the reaction at 70°C for 15 minutes (spin at 1400rpm for 15sec and stand for 2min). For second strand cDNA fabrication, 10mM dNTPs (NEB), 200U of E. coli DNA PolymeraseⅠ(Invitrogen) and 4U of E. coli RNase H (Invitrogen) were mix in the tube to a total volume of 150 μl; this reaction mix was incubated at 16℃ for 2.5 hours in a thermomixer (spin at 1400rpm for 15sec and stand for 2min). lastly, beads with cDNA was suspended in 1×NEBuffer 4 (NEB). The cDNA covalent linked to dynalbeads was digested using 10U of NlaⅢ (NEB) by incubating at 37℃ for 1 hours (spin at 1400rpm for 15sec and stand for 2min). Then, the cDNA was ligated to NlaⅢ adapter 1 by 5U of T4 DNA ligase (Invitrogen), and the reaction was processed at 20℃ for 2 hours in a thermomixer spin at 1400rpm sustainedly. Sequentially, 8U of MmeⅠ(NEB) was added to digest cDNA into 17bp｡ｫ19bp fragment, the procedure was done in a thermomixer spin at 1400rpm sustainedly at 37℃ for 1.5 hours. Then, the supernatant was transferred into a new tube, and CIAP (Invitrogen) was introduced to dephosphorylate for 1 hr at 37°C. After purification by Phenol/chloroform/isoamyl alcohol 25:24:1 (Invitrogen), the cDNA fragment was deposited by 100% ethanol combined with glycogen (Ambion) and acetate sodium (Novagen) and resolved in ultra pure water. The eluted cDNA was ligated to GEX adapter 2 utilized 5U of T4 DNA ligase (Invitrogen) by incubate at 20°C for 2 hours without any vibration. Subsequently, the cDNA fragment was amplified by 15 cycles PCR. The composition of liquid system and thermo cycling steps were the same as cDNA library procedure except the primer using GEX PCR primer 1 and GEX PCR primer 2 replace of primer1.1 and primer2.1. The products were purified by electrophoresis with 6% Novex™ TBE gel (Invitrogen) and ｡ｫ85nt DNA band on gel was separated and recovered. Then, we utilized 1×NEbuffer 2 (NEB) rotating at room temperature for 2 hours to facilitate elution and used Spin-X Cellulose Acetate filters (Fisher) to separate gel debris. Afterward, DNA was precipitated using 100% ethanol combined with glycogen (Ambion) and acetate sodium (Novagen). All oligonucleotides were provided by Illumina. Finally, the library products were sequenced using the 1G Illumina Genome Analyzer.
Data processing
21 bp short tags consisting of CATG and 17bp tag sequence were extracted from total reads and then adaptor tag ,low quality tag(containing Ns) were removed
