Rnaeasy mini clean up kit

Total RNA was extracted using the TRIzol reagent (Life Technologies, Grand Island, NY), followed by purification using the RNAeasy mini clean up kit (Qiagen, Valencia, CA). Tissue samples were extracted individually, RNA from leaf, pod and root was then pooled in equimolar amounts and submitted for sequencing. The quality and quantity of RNA were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Complementary DNA libraries were prepared and bar-coded for each of these accessions at the National Center for Genome Resources. RNA sequencing was performed on a GAIIx Analyzer (Illumina, San Diego, CA) for the tetraploid, and on a HiSeq 2000 (Illumina, San Diego, CA) for diploids.

Tissue samples for RNA extraction were obtained from five seedlings pooled together per replicate. Two replicates were extracted separately using the TRIzol reagent and company recommended protocols (Life Technologies, Grand Island, NY). The total RNA samples from each replicate were quantified using Nano Drop and subsequently purified using the RNAeasy mini clean up kit (Qiagen, Valencia, CA). The quality and quantity of RNA were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Subsequent cDNA library construction and bar-coding for each of the samples from the two genotypes under each temperature treatment was conducted at the National Center for Genome Resources. RNA sequencing was performed on a GAIIx Analyzer (Illumina, San Diego, CA).

For this study 20 seeds for each of the cultivar Hong Ke Zi (cold tolerant) and BTx623 (cold sensitive) were planted in ragdoll set up under cold stress temperatures. The temperatures were continuous 14 °C for cold stress using Conviron walk-in growth cabinets. The seedlings were allowed to grow under 14 h light/10 h dark conditions. Leaf tissue samples for RNA extraction were obtained from five seedlings per replicate for Hong Ke Zi and BTx623. Three replicates were extracted separately using the TRIzol reagent and company recommended protocols (Life Technologies, Grand Island, NY). The total RNA samples from each replicate were quantified using Nano Drop and subsequently purified using the RNAeasy mini clean up kit (Qiagen, Valencia, CA). To evaluate the expression differences observed, qRT-PCR primers were designed for four selected genes in the expression module using primer3 (Additional file 7) and sorghum actin gene was used for normalization. Briefly, qRT-PCR was performed using copy-DNA libraries generated from the total RNA using Invitrogen cDNA synthesis kit (Invitrogen, Grand Island, NY). qRT-PCR was performed on the cDNA of the four samples with 3 biological and 3 technical replicates using SybrGreen on LightCycler 480.