Exchange of molecular and cellular information: a hybrid model that integrates stem cell divisions and key regulatory interactions [w5C6]

We performed a transcriptome analysis on FACS-sorted GFP positive cells from pCYCD6;1-GFP, wox5xpCYCD6;1-GFP, and pWOX5-GFP, and the GFP negative cells from pWOX5-GFP to obtain CEI specific transcriptional profiles in a WT and in a wox5 mutant background. Overall design: Four biological replicates of pWOX5:erGFP, pCYCD6:GUS-GFP, and wox5 x pCYCD6:GUS-GFP seeds were plated. After 5 days of growth, approximately 1mm of the root tip was collected and protoplasted as described (Birnbaum et al., 2005). GFP positive and negative cells were collected using a MoFlo cell sorter into a vial containing a solution of beta-mercaptoethanol and RLT buffer. RNA was extracted using the Qiagen RNeasy Micro kit. Libraries were prepared using the SMART-Seq v3 Ultra Low RNA Input Kit for Sequencing and the Low Library Prep Kit v1 from Clontech. Libraries were sequenced on an Illumina HiSeq 2500 with 100 bp single-end reads. Gene expression analysis of raw RNA-seq data was performed using the TuxNet interface (Spurney et al., 2019). Specifically, TuxNet uses ea-utils fastq-mcf (Aronesty, 2011, Aronesty, 2013) for preprocessing, hisat2 (Kim et al., 2015) for genome alignment, and Cufflinks (Trapnell et al., 2012) for differential expression analysis.