title: RNA-seq transcriptome profiling of upland cotton (Gossypium hirsutum cv. Siokra L-23) root tissue under water deficit stress
design: Root tissues from G. hirsutum cultivar Siokra L-23, a cultivar selected for its previously established high level of drought tolerance (Nepomuceno et al. 1998; Voloudakis et al., 2002; Stiller et al. 2005), were collected from field grown plants according to the method described by Park et al., 2012. Samples were harvested during the third week of flowering on a single sample date, when significant differences in xylem water potential of the uppermost fully expanded leaves between treatments occurred, as measured by a pressure bomb (Model 600, PMS Instrument Company, Albany, OR). Average water potential of leaf tissue from well watered and water deficit stressed plants can be found in Table 1. Total RNA was isolated as previously described (Park et al., 2010, Park et al., 2012) using the XT buffer system with the addition of chloroform/iso-amyl alcohol extraction and LiCl precipitation steps (Wan, Ching-Yi, Wilkins 1994).     RNA-Seq library Construction and Sequencing  Barcoded libraries were created with the Illumina RNA TruSeq kit (Illumina) as per manufacturer's instructions for three individual root RNA samples for each treatment (How much RNA?). Library quality was assessed on an Agilent Bioanalyzer 2100 (Agilent) and the concentration of each individual library was calculated using qPCR (What machine? Anything else to add here?).  Libraries were pooled together so that each barcode was represented in equimolar amounts and sequenced in a single lane of 50bp Illumina HiSeq 2000.     Read Trimming and Mapping  Reads were trimmed with Sickle (https://github.com/najoshi/sickle) with a quality cutoff of 20. Genomic Short-Read Nucleotide Alignment Program (GSNAP) (Wu and Nacu 2010) was used to map reads to the G. raimondii 2.1 whole genome reference sequence (Paterson et al. 2012), with SNP-tolerant mapping using a SNP-index based on deep coverage of G. arboreum and G. raimondii, as described in Page et al., 2013. The "-N 1" option was used for GSNAP to identify novel splice sites. Putative A and D sub-genome localization of the differentially expressed transcripts was conducted using the PolyCat pipeline (Page et al., 2013), which categorizes and maps DNA sequence reads of allotetraploid G. hirsutum to the A and D progenitor genomes G. arboreum (A2) and G. raimondii (D5).