Illumina HiSeq 2000 sequencing; RNA-Seq of Bovan brown chicken posterior forewing regions from control and stage HH19 cyclopamine-treated samples, posterior foreleg regions of control Bovan brown and feathered Pekin bantams collected at E10.5
RNA-Seq of Bovan brown chicken posterior forewing regions from control and stage HH19 cyclopamine-treated samples, posterior foreleg regions of control Bovan brown and feathered Pekin bantams collected at E10.5
Workspace and dissection tools were thoroughly cleaned using RNaseZap (Ambion), then rinsed in diethyl pyrocarbonate (depc, SIGMA) treated water. Embryos at the desired stage were dissected into fresh depc treated 1xPBS (Phosphate buffered saline). Posterior forewing/foreleg sections were cut out from the embryo using micro-dissection scissors and placed in RNAlater and stored at -80°C (Ambion). Posterior forelimb sections were collected from embryonic chickens at embryonic day 10.5 E10.5 (If cyclopmaine treated, this was carried out at Hamburger Hamilton Stage 19, E4.5). Samples were collected in triplicate (with 10 posterior limb sections pooled per replicate). Pb sample tissue was collected from Pekin bantam feathered leg chickens. All other samples are of a wild-type background (Bovans Brown chicken eggs). Addition of 5ul cyclopamine at Hamburger Hamilton stage HH19. Samples were homogenised in 1ml of Trizol. An equal volume of 100% ethanol was added and samples were purified using Zymo Research Direct-zol RNA Miniprep Kit. TruSeq Stranded Total RNA and Ribo-Zero. Enzymatic fragmentation by Illumina Elute, Fragment, Prime mix