Nugen ovation rna amplification system v2

Plants were grown on standard plates in standard conditions and transferred to CNQX or mock cutting board plates at 7 days after plating. Immediately after transfer, plants were cut at 270 µm (high cuts) and then transferred to CNQX plates or mock plates. Approximately 20 stump tips consisting of about 100 µm of the stump were collected at each time point (30 min, 4 h, 14 h, 24 h). For controls, two separate sets of uncut roots were transferred to CNQX plates or mock plates and incubated for 14 h before collecting root tip tissue, which also consisted of about 100 µm of the distal root tip. RNA was extracted using the Qiagen MicroRNAeasy Kit. We performed cDNA construction and library amplification with Nugen OvationRNA Amplification System V2. Libraries were made using the Nugen Ovation Ultralow DR Multiplex System (Tecan). DNA fragmenting was performed using the Covaris S220. Short read sequences were generated on an Illumina NextSeq 500 and were aligned using HISAT2. Counts were normalized between samples using DESeq2’s median of ratios method before analyzing for differentially expressed genes.

RNA was isolated using the QIAGEN^® RNeasy micro kit (QIAGEN, Santa Clarita, CA). Isolated RNA was then sent to the UCLA Clinical Microarray Core (CMC) for subsequent processing. The samples were amplified using the NuGEN Ovation RNA Amplification System V2 (cat. no. 3100-A01; NuGEN Technologies Inc., San Carlos, CA) and were then hybridized to Affymetrix Mouse Genome 430 2.0 Array. Affymetrix GeneChip^® CEL files for the samples assayed were imported into the Affymetrix Expression Console^™ software to perform gene level normalization and signal summarizations. Affymetrix Transcriptome Analysis Console (TAC) software was used to analyze the differential gene expression between the irradiation time points. The Affymetrix TAC software computed the fold changes, ANOVA and FDR P value across all conditions for all the probe sets for each of the pairwise conditions. A comparison between no irradiation and 6 h postirradiation was used for the heat map analysis in Fig. 2. Genes that demonstrated a greater than tenfold change and FDR P value greater than 0.05 were used for the heat map analysis. Robust multi-array average (RMA) values generated in the Affymetrix Expression Console software from this gene list were imported into Partek^® microarray data analysis software (Partek Inc., St. Louis, MO) and unsupervised hierarchical cluster was performed on the various gene lists.