title: RNA-Seq of Quercus lobata: MC8-8 leaf before water stress
design: Acorns were planted in Sunshine #4 aggregate plus soil mix in Steuwe and Sons D40 pots (25 cm depth _ 6.4 cm diameter) November 2011 and watered twice per week and as needed to maintain moist soil. On 23 August 2012, mature leaf tissue samples (fourth node from top) were collected and frozen immediately between slabs of dry ice for RNA isolation to be used as control samples before treatment (ŇbeforeÓ samples). To simulate a water stress condition, these same seedlings were deprived of water for 15 days, after which (7 September 2012) we collected tissue on dry ice for RNA isolation (ŇafterÓ samples). Total RNA was isolated with a prewash protocol (http://openwetware.org/wiki/Conifer_RNA_prep) and Qiagen RNeasy Mini Kit. The quality and quantity of the total RNA was determined using an Agilent BioAnalyzer 2100 with RNA Nano chip. We then prepared cDNA from poly-A purified mRNA for 50-bp, single-end sequencing using the Illumina TruSeq RNA library preparation kit. Each sample was barcoded using standard Illumina adaptors 1 Đ 12 to allow up to 12 samples to be pooled in each of two lanes of sequencing on an Illumina HiSeq 2000 v3 at the Broad Stem Cell Research Center, UCLA. A given before-after pair was sequenced on the same lane, and sample pairs drawn from different populations were stratified across the two sequencing lanes. Samples were demultiplexed allowing up to one mismatch with the expected barcode sequence and converted to FASTQ format.