title: Barley guard cell and subsidiary cell specific transcripts
design: Barley guard cell and subsidiary cell specific transcripts
library_construction_protocol: Epidermal peels were collected from the abaxial side of 8 to 10-day-old leaves. To prepare isolated epidermal peels, leaves were cut from the plant and bent over the forefinger with the adaxial surface facing upward. A shallow cut was made with a sharp razor blade horizontally across the leaf and a flap of leaf tissue lifted with a razor, leaving the lower epidermis intact. The leaf tissue was removed from the epidermis with forceps. Samples were taken from whole leaves epidermal peels and highly GC enriched tissue. Epidermal peels from 12-day-old Barley (cv. Barke) leaves were isolated. Thereby only the GC SC complex survives. For GC samples we used the 'blender method' on epidermal peels with mature, intact GCs to mechanically and selectively destroy the SCs while keeping GCs alive. GC were enriched within 8 minutes by successive blender cycles (45 s each) in ice-cold deionized water with additional crushed ice and filtered through a 210-mm nylon mash. After two rounds of blending, the remaining light green epidermal fraction was further processed. Neutral red staining indicated that at least 90% of the viable cells in the preparations were GCs. Barley (Hordeum vulgare cv. Barke) seeds were provided by a commercial supplier (Saatzucht J. Breun GmbH & Co. KG) and cultivated at 22/16C and 50 ± 5% RH at a 12/12h day/night cycle and a photon flux density of 500 mmol m-2 s-1 white light (Philips Master T Green Powers, 400 W). RNA was extracted from a total of 20 epidermal peels per sample using the NucleoSpin RNA Plant Kit (Macherey-Nagel,Drueren, Germany). RNA isolation from whole leaves was performed similarly. The extracted RNA was treated with RNase-free DNase (New England Biolabs, Ipswich, MA, USA). Quality control measurements were performed on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Libraries were prepared with the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) using 1 mg of RNA.