title: Blackface Teladorsagia Solexa
design: Blackface Teladorsagia Solexa
library_construction_protocol: Uninfected control lambs, were sham dosed with water at the same time as three times a week starting at approximately 12 weeks of age and continuing until sacrifice at approximately 27 weeks. Gastric lymph nodes were collected from all lambs, diced and placed in RNAlater (Ambion) at 20C, incubated at 4C overnight and stored at -70C. Total RNA was isolated from tissues using the Ambion Ribopure (http://www.ambion.com/techlib/prot/fm_1924.pdf). Approximately 100 mg tissue was thawed to 20C and excess RNALater removed by blotting. 2 ml TRI Reagent was added and tissues homogenized using a rotor-stator homogenizer. Insoluble material was removed by centrifugation at 12,000 x g. 1 ml  supernatant from each sample was dispensed into 1.5 ml microcentrifuge tubes with 100 ul of bromochloropropane (BCP). Tubes were vortexed and incubated at 20C for 15 min. Tubes were centrifuged at 12,000 x g for 10 min at 4C. 400 ul of the aqueous phase (top layer) was transferred to a new 1.5 ml microcentrifuge tube with 200 ul 100% ethanol, vortexed for 5 s. Each sample was transferred to a Filter Cartridge-Collection Tube assembly, centrifuged at 12,000 x g for 30 s at 20C. 500 ul of Wash Solution was added to the Filter Cartridge-Collection Tube centrifuged for 30 s at 20C and rewashed. RNA was eluted by the addition of 100 ul Elution Buffer incubated at 20C for 2 min and centrifuged for 30 s to elute the RNA from the filter. RNA quality and integrity was assessed using a RNA 6000 Nano LabChip on the Agilent 2100 Bioanalyzer and quantified using a NanoDrop ND-1000 spectrophotometer.
attributes.tag: Experimental Factor: CLINICALINFORMATION
attributes.value: not applicable
attributes.tag: Experimental Factor: GROWTHCONDITION
attributes.value: uninfected control