title: HH29 a
organism: Gallus gallus
description: Protocols: 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
attributes.tag: ENA first public
attributes.value: 2018-04-20
attributes.tag: ENA last update
attributes.value: 2018-01-31
attributes.tag: External Id
attributes.value: SAMEA104556989
attributes.tag: INSDC center alias
attributes.value: The University of Sheffield
attributes.tag: INSDC center name
attributes.value: The University of Sheffield
attributes.tag: INSDC first public
attributes.value: 2018-04-20T17:03:23Z
attributes.tag: INSDC last update
attributes.value: 2018-01-31T12:02:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6437:HH29 a
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: mesenchymal stem cell
attributes.tag: common name
attributes.value: chicken
attributes.tag: developmental stage
attributes.value: Hamburger Hamilton Stage 29
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: distal limb tip
attributes.tag: sample name
attributes.value: E-MTAB-6437:HH29 a