Acid phenol chloroform mb grade

Stem bark tissue (~ 2 g) from 29-month old guayule plants was the source of RNA for sequencing and qPCR analysis. Total RNA was extracted following Laudencia et al.^{98 (link)} protocol with the use of acid phenol:chloroform MB grade (Ambion, USA) instead of phenol:chloroform:isoamyl alcohol. The precipitated RNA was further cleaned with Qiagen RNeasy Plant Mini Kit (Qiagen, USA) and treated with DNA-free™ kit (Ambion, USA). PolyA-RNA was prepared employing Qiagen RNeasy/QIAshredder protocols (Qiagen, USA). RNA concentration was quantified with Quant-iT™ RiboGreen™ RNA Assay Kit (Thermofisher Scientific, USA). RNA quality was analyzed using 2100 Bioanalyzer (Agilent Technologies, USA). RNAseq library construction was carried out with KAPA Stranded RNA-Seq Library Preparation Kit Illumina® platforms (Kapa Biosystems, USA) per the manufacturer's instructions. RNAseq libraries with insert sizes of 200–500 bp, and sequenced using Illumina HiSeq 2000 platform with paired-end (PE) reads of 150 bp.

Approximately 2 g of stem tissue was used for total RNA extracted following the Laudencia et al. 2007 (50 (link)) protocol with the following modifications: (i) acid phenol:chloroform MB grade (Ambion, USA) was used for the phenol:chloroform extraction step; (ii) the precipitated RNA was further cleaned with Qiagen RNeasy Plant Mini Kit (Qiagen, USA); and (iii) the cleaned RNA was treated with DNA-free™ kit (Ambion, USA). PolyA-RNA was prepared employing Qiagen RNeasy/QIAshredder protocols (Qiagen, USA). RNA-sequencing libraries were prepared using the KAPA stranded mRNA-seq kit for Illumina (KK8420) according to the manufacturer’s protocol (KR0960 - v3.15). RNA-sequencing was performed on the Illumina HiSeq2000 with 150 bp paired-end reads. A total of 98,430,986 reads was generated for the six samples.