characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-01-09T17:01:37Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T22:07:16Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA3158531
characteristics.characteristic: External Id
characteristics.text: University of Dundee
characteristics.characteristic: INSDC center name
characteristics.text: 2015-01-09T17:01:37Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T22:07:16Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS620483
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-3135:Kc167_RpL22_IP
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Kc167
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005819
characteristics.characteristic: cell line
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were cultured in the presence of 10% FBS in DMEM (U2OS/Hela cells) or in Schneider's medium (Kc167 cells). Both cell types were stimulated with 10 % extra FBS for 1h followed by 250 uM cycloheximide for 10 min. For ribosome profiling of Drosophila RpS8, RpL13 and RpL22 ORFs tagged with O6-alkylguanine-DNA-alkyltransferase (AGT)-2xprotein G tag to the C terminus of the ORF., 30 ml of each culture were lysed and digested as before with MN (200 U). Digestion was stopped with EGTA and 350ul goat anti-rabbit IgG magnetic bead suspension (New England BioLabs) together with SUPERase-In (1/500) were added to the lysates and incubated o/n at 4 degrees C with gentle mixing. The beads were collected and washed three times with the lysis buffer. RNA was extracted from the beads using 500ul Qiazol reagent and the ribosome profiling was continued as before. 5-15 picomoles of each RNA sample was used for phosphatase and poly-A tailing reaction by mixing 3.25ul of RNA with the following: 0.5ul 10x poly-A buffer (0.5 M Tris-HCl, pH 7.0, 2.5 M NaCl, 100 mM MgCl2, 50 mM DTT, 1 mg/ml BSA), 0.25ul Yeast poly-A polymerase (600 U/ul, Affymetrix), 0.5ul 10 mM ATP, 0.3ul (3 U) T4 polynucleotide kinase (ThermoFisher), 0.2ul SUPERase-In. Samples were incubated at 37 degrees C for 3 h. Note that RNase I digestion results in cyclic 2'3'-phosphate, which will be resolved by the inherent phosphatase activity of the T4 polynucleotide kinase. 4ul of each sample was mixed with 1ul 10uM abasic-oligo dT primers containing random 8 nt sequence used for absolute molecular counting (Casbon et al., 2011; Kivioja et al., 2012), incubated at 72 degrees C for 3 min and put on ice. Reverse transcription was started by adding the following to the samples: 5.25ul H20, 3ul 5x RT-buffer (ThermoFisher), 0.75ul 10 mM dNTP, 0.25ul SUPERase-In and 0.4ul Maxima Reverse transcriptase (75 U, ThermoFisher). Samples were incubated at 50 degrees C for 1 h, heat inactivated at 85 degrees C for 6 min after which 0.5ul RNAse H (ThermoFisher) was added and samples were incubated at 37 degrees C for 30 min. Reverse transcription products were purified on 10% TBE-urea gel and cDNA was eluted into 700ul of DNA gel elution buffer (1x TE + 300 mM NaCl) by incubating o/n at 4 degrees C with mixing on Eppendorf Thermomixer. cDNA was precipitated as before, samples were centrifuged for 10 min at 16000 g, pellets were washed and cDNA was suspended into 5ul of circularization mix (0.5ul CircLigase buffer (10x), 0.25ul 50 mM MnCl2, 0.25ul CircLigase II (Epicentre), 1ul 5 M betaine, 3ul sterile water). Samples were incubated o/n at 60 degrees C and heat-inactivate for 10 min at 80 degrees C. cDNA was linearized by adding 3ul of relinearization supplement (50 mM KCl, 1 mM DTT) followed by 0.7ul (7 U) APE1 (New England Biolabs). Samples were incubated for 1 hour at 37 degrees C. Libraries were then amplified using 18 cycles of PCR with Solexa-primer-F and R and Phusion DNA polymerase (ThermoFisher). PCR products were separated on agarose gel, eluated using Qiagen MinElute Gel extraction kit and quantified with Quant-it PicoGreen DNA dye (LifeTechnologies) using Qubit fluorometer (LifeTechnologies).
characteristics.characteristic: description
characteristics.text: tagged RpL22 expression
characteristics.characteristic: genotype
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: Drosophila melanogaster
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_7227
characteristics.characteristic: organism
characteristics.text: control conditions with micrococcal nuclease
characteristics.characteristic: protocol
characteristics.text: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: Kc167_RpL22_IP
characteristics.characteristic: title