GSM8345881: Root,  14-B20_S11; Arabidopsis thaliana; RNA-Seq
Total RNA from frozen root samples was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA concentration, integrity, and quality were determined with a Qubit RNA Broad-Range Assay Kit (Invitrogen, Waltham, MA, USA) and a Qubit 4 benchtop fluorometer (Invitrogen, Waltham, MA, USA). RNA integrity was further assessed with a 2100 Bioanalyzer mRNA sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core (https://rtsf. natsci.msu.edu/genomics/). Libraries were prepared using the Illumina Stranded mRNA Library Preparation, Ligation Kit with IDT for Illumina Unique Dual Index adapters following the manufacturer's recommendations except that half volume reactions were used. Completed libraries were quality checked and quantified using a combination of Qubit dsDNA high sensitivity (HS) and Agilent 4200 Tape Station HS DNA1000 assays. The libraries were pooled in equimolar proportions and the pool quantified using the Invitrogen Collibri Quantification qPCR kit. The pool was combined with other pools of Illumina Stranded RNA libraries prepared by the Genomics Core to make use of a shared S4 lane. This combined pool was likewise quantified using the Invitrogen Collibri Quantification qPCR kit. The combined pool was loaded onto one lane of an Illumina S4 flow cell and sequencing was performed in a 2x150 bp paired end format using a NovaSeq 6000 v1.5, 300 cycle reagent kit. Base calling was done by Illumina Real Time Analysis (RTA) v3.4.4 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0.