RNA-seq of tomato under low temperature
The total RNAs were isolated from leaves of tomato seedlings using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturers instruction. The concentration and purity of total RNAs were assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Ribosomal RNA (rRNA) was removed by Ribo-Zero rRNA Removal Kits (Illumina, San Diego, USA). The RNA integrity was assayed on an Agilent 2100 Bioanalyzer Lab-on-Chip system (Agilent Technologies, Palo Alto, CA, USA). The rRNA-depleted RNAs were further incubated at 37 C for 1 hour in 16 l reaction with 10U/g RNase R (Epicentre, Madison, WI). The remaining RNAs were used as templates for the construction of cDNA libraries in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA). The clustering of samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, USA) following the manufacturers procedure. The paired-end sequencing was performed on the Illumina HiseqX Ten platform.