Barley Flag Leaf Transcriptome Analysis

Flag leaves of barley genotypes were collected from all experimental variants at two time points (T1 and T2), and they were immediately frozen in liquid nitrogen and stored at −80°C until analysis. Total RNA (1–2 µg) was extracted in triplicates using the Qiagen (RRID : SCR_008539, http://www.qiagen.com, Hilden, Germany) RNeasy Plant Mini Kit according to the manufacturer’s instructions. Genomic DNA contamination was removed twice, i.e., on-column DNase digestion (RNase-Free DNase Set, Qiagen) and using the DNase Max Kit (Qiagen) during and after RNA isolation, respectively. Three flag leaves sampled from different plants of each genotype in a pot formed one replication, and three such replications were examined. RNA quantity, quality, and integrity were evaluated following the study by Mikołajczak et al. (2022) (link). The construction of a cDNA library (TruSeq stranded mRNA) and mRNA sequencing were performed by Macrogen Inc. (RRID : SCR_014454, http://www.macrogen.com, Seoul, Republic of Korea) using an Illumina NovaSeq6000 platform with a 150-bp paired-end configuration and the number of read pairs ranging from 18.3 to 40.9 M per sample.

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Mikołajczak K., Kuczyńska A., Krajewski P., Kempa M, & Nuc M. (2023). Transcriptome profiling disclosed the effect of single and combined drought and heat stress on reprogramming of genes expression in barley flag leaf. Frontiers in Plant Science, 13, 1096685.

Publication 2022

RNeasy Plant Mini Kit RNase-Free DNase Set DNase Max Kit NovaSeq6000 platform

Barley Cdna library Digestion Dna contamination Dnase Frozen Genomic Genotype Isolation Mrna Nitrogen Plants Replications Rnase

Corresponding Organization :

Other organizations : Institute of Plant Genetics, Polish Academy of Sciences, Polish Academy of Sciences

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Variable analysis

independent variables

Barley genotypes

dependent variables

Total RNA quantity, quality, and integrity
MRNA sequencing (number of read pairs)

control variables

Time points (T1 and T2)
Liquid nitrogen freezing and storage at -80°C
RNA extraction using Qiagen RNeasy Plant Mini Kit
Genomic DNA contamination removal using on-column DNase digestion and DNase Max Kit
RNA quantity, quality, and integrity evaluation following Mikołajczak et al. (2022) study
CDNA library construction (TruSeq stranded mRNA) and mRNA sequencing by Macrogen Inc. using Illumina NovaSeq6000 platform

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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