README_Wheat RNA-Seq Processing Procedure.txt

1. The raw RNA-seq reads (available at GEO GSE137895) were preprocessed by trimming the adaptor sequences (adaptor info provided in github), filtering low-quality reads (Phred Score ≤ 20) and eliminating short reads (length ≤ 20 bps) using software package FASTX-toolkit, in which "fastx_clipper" was used to remove adaptors, and "fastq_quality_trimmer" was used to remove low quality and short reads.
2. The cleaned RNA-seq reads in each sample were mapped using STAR to generate gene-level counts. 
   First following command was used for generating index, "STAR --runThreadN 32 --runMode genomeGenerate --genomeDir ${reference_index_dir} --genomeFastaFiles ${reference_GenomeFasta_dir} --sjdbGTFfile ${gtf_dir} --sjdbOverhang ${overhangLength} --limitGenomeGenerateRAM 100000000000 --genomeSAindexNbases ${myGenomeSAindexNbases}", 
   then following command was used for the alignment: "STAR --sysShell /bin/bash --runMode alignReads --runThreadN 32 --limitBAMsortRAM 100000000000 --limitIObufferSize 500000000 --limitSjdbInsertNsj 5000000 --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --outSAMmode Full --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --chimSegmentMin 20 --quantMode TranscriptomeSAM GeneCounts --outBAMsortingThreadN 0 --outSAMattributes All --outWigType None --genomeDir {reference_index_dir} --readFilesIn reads1 reads2 --outFileNamePrefix output_dir
3. Extract alignment results of each sample to make a text file, each column is the aligned read count for the genome
4. Then run the R code (provided in github) using the above combined read count files for differential expression analysis using DEseq2.