E-MTAB-6437:HH27 c_p
Illumina HiSeq 2000 paired end sequencing; RNA sequencing of chicken limb distal mesenchyme cells to compare gene expression over time and in heterochronic grafted cells
RNA sequencing of chicken limb distal mesenchyme cells to compare gene expression over time and in heterochronic grafted cells
HH27 c_p
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). Distal limb tips (250μm in length) were cut out from the embryo using micro-dissection scissors and placed in RNAlater and stored at -20°C (Ambion). Each sample consists of 10 distal tips pooled together. Distal limb mesenchyme cells were collected from embryonic chickens at Hamburger Hamilton (HH) stages HH24, HH27, and HH29, and also from samples that had been grafted from HH27 to HH20 and left to develop for 24 hours (equivalent to HH24). Samples were collected in triplicate (with 10 distal tips pooled per replicate). Homogenisation: Add 1ml of Trizol and homogenise in an eppendorf. Sample incubated at room temperature for 5 minutes. 0.2 ml chloroform added, and sample shaken vigorously by hand for 15 seconds. Incubate at room temperature for 2 - 3 minutes and centrifuge samples at 12000 x g for 15 minutes at 4 °C. Transfer aqueous (upper) phase to a new tube. Add 1 volume of 70% Ethanol, and then follow protocol as per the PureLink RNA Mini kit (Ambion). Resuspend in 30μl RNAse free water. TruSeq Stranded Total RNA and Ribo-Zero. Enzymatic fragmentation by Illumina Elute, Fragment, Prime mix
The University of Sheffield
E-MTAB-6437:HH27 c_p
Experimental Factor: developmental stage
Hamburger Hamilton Stage 27
Experimental Factor: growth condition
not applicable