Ambion purelink rna mini kit

Genomic DNA was extracted using direct PCR Lysis Reagent Cell (VWR) for PCR analysis or using Qiagen Blood and Tissue DNA isolation kit for WES and copy number analysis. RNA isolation was performed using the AMBION PureLink RNA Mini kit (Themo Fisher) according to the manufacturer’s instruction, including an on-column DNase digest. RNA was reversely transcribed using gene-specific primer and a two-step protocol. PCR amplifications were performed using Phusion polymerase (New England Biolabs), products were analyzed by agarose gel electrophoresis and detected using RedSafe/UV light. Primer sequences are listed in Supplementary Table 4. Of note, additional low mobility bands were observed from PCR of one-allelic knock-in clones (Supplementary Figs. 1h and 6c). Sanger sequencing of one of those bands revealed a mixture of wild-type and knock-in sequence. This and the fact that we did not observe low mobility bands in PCRs from bi-allelic knock-in clones made us conclude that they represent slowly migrating heteroduplexes and not additional PCR products.

For transcriptome sequencing, total RNA was extracted from three replicates (six plants each) for each treatment using the Trizol method, and purification was carried out using the Ambion PureLink RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. mRNA was prepared by LGC Genomics using oligo (dT) magnetic beads, followed by cDNA synthesis. Sequencing libraries were prepared using the Ovation Rapid Library Preparation kit (NuGEN, San Carlos, CA, USA). The libraries were sequenced using the Illumina HiSeq 4000 platform (Illumina Inc., San Diego, CA, USA) to obtain 75 bp-long single-end reads. For data analysis, sequencing adaptors were trimmed from raw reads using cutadapt and reads with final lengths of <20 were discarded. Ribosomal RNA contamination was removed using SortMeRNA (v2.1) [101 (link)], and reads aligning to rRNA were filtered out. Filtered reads for all samples were quantified using kallisto (v0.43.0; bootstraps: 100) [102 (link)] against Arabidopsis cDNA sequences (Araport11) [103 (link)]. Differential expression analysis was carried out using the EdgeR package in R/Bioconductor [104 (link)]. Significantly differentially expressed genes were identified using log2 fold change ≥1 and FDR cut-off of <0.001. GO enrichment was carried out using the GOSeq R-package [105 (link)], with FDR cut-off of ≤0.1.