title: Illumina HiSeq 2000 paired end sequencing; A role for differential gene regulation in the rapid diversification of melanic plumage coloration in the dark-eyed junco (Junco hyemalis)
design: A role for differential gene regulation in the rapid diversification of melanic plumage coloration in the dark-eyed junco (Junco hyemalis)
library_construction_protocol: We sampled feathers from two dark-eyed junco (Junco hyemalis) subspecies: the Oregon junco (n=4) and the slate-colored junco (n=4). Oregon juncos were originally captured on University of California San Diego campus and the Laguna Mountains in the San Diego County, California, USA. Slate-colored juncos were captured at Mountain Lake Biological Station, Giles County, Virginia, USA. In order to reduce the influence of external factors on gene expression, birds were kept in a common-garden environment for at least a year previous to the collection of feathers. All birds used in the experiment were placed in cages in a single room, under similar light and temperature conditions, and were fed the same food. For each individual, we plucked mature feathers from four distinct body areas: head (coronal region of the capital feather tract), back (interscapular region of the spinal tract), flank (dorsal side of the sternal region of the ventral tract), and ventral area (belly; the ventral side of the sternal region of the ventral feather tract). For each tract, we plucked feathers from area of 1 cm2. The mature feathers were used for the pigment composition and distribution analysis, while the plucking served to induce feather development in the plucked area. We monitored feather regrowth every two days following plucking. We collected developing feathers when the first mature barbs started to erupt from the tip of the follicle (8 to 19 days following the plucking, median 11 days). To collect feather follicles, we applied a topical anesthetic to the skin, and gently plucked individual developing feathers using forceps. Six follicles were collected from each area. Plucked follicles were immediately placed on pulverized dry ice, and thereafter frozen at -80 ºC until RNA extraction To analyze gene expression, we pooled 6 developing feathers from each of four body areas (head, back, flank, ventral) for each of four individuals per morph (total number of libraries was 32). RNA was extracted in TRIzol following manufacturer directions (Invitrogen, Carlsbad, CA, USA) cDNA libraries were prepared at Macrogen Inc., South Korea, using Illumina Truseq RNA technology