title: MtCBF4 regulation genes in Medicago truncatula-35S:MtCBF4
design: A total amount of 3 μg RNA per sample was used as input material for the RNA sample  preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library  Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index  codes were added to attribute sequences to each sample. Briefly, mRNA was purified from  total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using  divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction  Buffer （ 5X ） . First strand cDNA was synthesized using random hexamer primer and  M-MuLV Reverse Transcriptase （ RNase H- ） . Second strand cDNA synthesis was  subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were  converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’  ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to  prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp  in length, the library fragments were purified with AMPure XP system (Beckman Coulter,  Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected,  adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then  PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers  and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library  quality was assessed on the Agilent Bioanalyzer 2100 system.