RNA-seq analysis of barley hvcbp20.ab, hvcbp80.b and hvcbp20.ab/hvcbp80.b under optimal conditions
We used as an object barley plants (Hordeum vulgare (L.)), cultivar 'Sebastian' and its mutants TILLING hvcbp20.ab, hvcbp80.b and hvcbp20.ab/hvcbp80.b. The plants were grown in optimal water content (15% volumetric water content). The soil moisture was monitored daily using the TDR EasyTest (Institute of Agrophysics, Polish Academy of Sciences, Poland). The tissue was collected from the 3rd barley leaf from its middle part in 4 biological replicates, where each replicate included tissue from 3 independent plants. High-quality reads were aligned to the barley reference transcriptome (BaRTv2.18) with Kallisto v. 0.46.2. Differential gene expression was analyzed using 3DRNAseq within the R software environment. Read counts and transcripts per million (TPM) were derived from Kallisto outputs via the tximport R package. Transcripts and genes with low expression levels were filtered based on the mean-variance relationship, and genes were classified as expressed if they met defined thresholds for counts per million (CPM). The trimmed mean of M-values (TMM) method was applied for count normalization, and principal component analysis (PCA) revealed no notable batch effects. We used as an object barley plants (Hordeum vulgare (L.)), cultivar 'Sebastian' and its mutants TILLING hvcbp20.ab, hvcbp80.b and hvcbp20.ab/hvcbp80.b. The plants were grown in optimal water content (15% volumetric water content). The soil moisture was monitored daily using the TDR EasyTest (Institute of Agrophysics, Polish Academy of Sciences, Poland). The tissue was collected from the 3rd barley leaf from its middle part in 4 biological replicates, where each replicate included tissue from 3 independent plants. High-quality reads were aligned to the barley reference transcriptome (BaRTv2.18) with Kallisto v. 0.46.2. Differential gene expression was analyzed using 3DRNAseq within the R software environment. Read counts and transcripts per million (TPM) were derived from Kallisto outputs via the tximport R package. Transcripts and genes with low expression levels were filtered based on the mean-variance relationship, and genes were classified as expressed if they met defined thresholds for counts per million (CPM). The trimmed mean of M-values (TMM) method was applied for count normalization, and principal component analysis (PCA) revealed no notable batch effects. Total RNA from leaf tissue was extracted using miRvana isolation kit (ThermoFisher Scientific, USA) according to the manufacturer's guidelines. Concentration and quality were assessed using a NanoDrop spectrophotometer (ND-1000). cDNA libraries generated using the Illumina TruSeq protocol were sequenced on an Illumina NovaSeq 6000 platform by Macrogen, producing paired-end reads of 2 × 150 bp length. Each sequencing lane contained six samples, and an entire flow cell was utilized. 