GSM8502979: maize,KN5585,rep2; Zea mays; RNA-Seq
The expression level were tested between maize inbred line KN5585 (WT) and  ZmPP2C45 knock-out line.  Leaves of two-week-old maize seedlings were used for RNAseq analyses Total RNA was used as input material for the RNA sample preparations. 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 First Strand Synthesis Reaction Buffer. 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, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified. Six gigabytes of Illumina PE150 raw reads were obtained for each sample. After filtering out adaptor and low-quality reads, the cleaned reads were mapped to the B73 v4 reference genome using the STAR 2.4.0j software After the library construction is completed, preliminary quantification is carried out using Qubit, and the library is diluted to 1.5ng/ul. Subsequently, the inserted fragments of the library are detected. Once the inserted fragments meet expectations, the effective concentration of the library is accurately quantified using qRT PCR to ensure the quality of the library. The qualified libraries were pooled and sequenced on sequencing platforms, according to effective library concentration and data amount required. The original fluorescence image files obtained from Illumina platform are transformed to short reads (Raw data) by base calling and these short reads are recorded in FASTQ format (Cock P. et al, 2010), which contains sequence information and corresponding sequencing quality information. Sequence artifacts, including reads containing adapter contamination, low-quality nucleotides and unrecognizable nucleotide (N), undoubtedly set the barrier for the subsequent reliable bioinformatics analysis. Hence quality control is an essential step and applied to guarantee the meaningful downstream analysis. we used Fastp (version 0.23.1)(Chen S. et al, 2018) to perform basic statistics on the quality of the raw reads.