Agilent 2100 bioanalyzer rna nano chip

Total RNA was extracted from these materials using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. The quality of total RNA was determined using a NanoDrop Spectrometer (ND-1000 Spectrophotometer, Peqlab). The mRNAs were isolated from total RNAs using the PolyATtract mRNA Isolation Systems kit (Promega) and condensed using the RNeasy RNA cleaning kit (Qiagen, Germany); their concentration and purity were determined using the Agilent 2100 Bioanalyzer (RNA Nano Chip, Agilent). The mRNAs were fragmented and retrieved using an RNA Fragment reagent kit (Illumina) and RNeasy RNA cleaning kit (Qiagen). Then, random primers and M-MLV were used to synthesize the first chain, and DNA Polymerase I and RNase H were used to synthesize the second chain. Finally, the cDNAs were retrieved using the RNeasy RNA cleaning kit (Qiagen, Germany), and their quality was checked using the Agilent 2100 Bioanalyzer. All procedures were performed according to the manufacturers’ instructions.

Total RNA was extracted from 14 week old whole roots, of colored carrot cultivars (B493B, R6637, Y9244A, P7262), with three roots (i.e. three biological replicates) per sample set. Total RNA was extracted using the TRIzol Plus RNA Purification Kit (Life Technologies, Carlsbad, CA) following the manufacturer’s protocol. DNA was removed with the ‘DNA free-kit’ provided with the RNA purification kit. RNA quantification was measured on a Nanodrop One Spectrophotometer and quality control was done on an Agilent 2100 Bioanalyzer RNA NanoChip. For each RNA sample, libraries were prepared at the University of Wisconsin-Madison Gene Expression Center and sequenced on an Illumina HiSeq 2000 using 1×100 nt reads. After quality control with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), reads were filtered with Trimmomatic version 0.32 with adapter trimming and using a sliding window of length ≥50 and Phred quality score ≥28^{31 (link)}. Reads were mapped against the carrot genome sequence (GenBank accession LNRQ01000000.1) using Bowtie2 (Langmead and Salzberg 2012)^{37 (link)}. Illumina reads were mapped against the carrot genome sequence (GenBank accession LNRQ01000000.1) using Rsubread version 1.24.2^{38 (link)}. Transcript expression was analyzed using the Bioconductor package limma^{39 (link)}.

Total RNA from GSG2 Gene knockdown cell model for microarray experiments was extracted by Trizol according to the manufacturer’s guidelines. RNA concentration and purity were quantified using a NanoDrop 2000 (Thremo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer RNA Nano Chip (Agilent Technologies, Palo Alto, CA, USA). Human GeneChip primeview (Affymetrix, Santa Clara, CA, USA) was used for microarray processing to determine gene expression according to the manufacturer’s instructions. RNA sequencing data processing and analysis were performed with R studio. Limma package was used for cluster analysis and differential expression of genes assessing. Canonical pathways, diseases and functions, molecular and cellular processes that are significantly associated with differentially expressed genes (DEGs) in the data sets were determined using Ingenuity Pathway Analysis (IPA) software. |Z score| > 2 is considered to be significant.