RNA-seq of Triticum aestivum
The library construction method and sequencing process are carried out according to the following steps:Take a certain amount of total RNA samples, and use oligo dT beads to enrich mRNA with poly A tail; 2. mRNA molecules were fragmented into small pieces; 3. The fragmented mRNA was synthesized into first strand cDNA using random primers; 4. The second strand cDNA was synthesized; 5. The synthesized cDNA was subjected to end-repair and 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 6. PCR amplification and purification; 7. Library quality control; 8. Library circularization; 9. The library was amplified to make DNA nanoball (DNB); 10. Sequencing on DNBSEQ (DNBSEQ Technology) platform. Raw data with adapter sequences or low-quality sequences was filtered. We first went through a series of data processing to remove contamination and obtain valid data. This step was completed by SOAPnuke software developed by BGI. SOAPnuke software filter parameters: " -n 0.01 -l 20 -q 0.4 --adaMR 0.25 --ada_trim --polyX 50 -- minReadLen 150", steps of filtering: 1. Filter adapter: if the sequencing read matches 25.0% or more of the adapter sequence (maximum 2 base mismatches are allowed), cut the adapter; 2. Filter read length: if the length of the sequencing read is less than 150 bp, discard the entire read; 3. Remove N: if the N content in the sequencing read accounts for 1.0% or more of the entire read, discard the entire read; 4. Remove polyX: if the length of polyX (X can be A, T, G or C) in the sequencing read exceeds 50bp, discard the entire read; 5. Filter low-quality data: if the bases with a quality value of less than 20 in the sequencing read account for 40.0% or more of the entire read, discard the entire read; 6. Obtain Clean reads: the output read quality value system is set to Phred+33.