title: Kc167_RpL13_IP
organism: Drosophila melanogaster
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-01-09T17:01:37Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T22:15:26Z
attributes.tag: External Id
attributes.value: SAMEA3158528
attributes.tag: INSDC center name
attributes.value: University of Dundee
attributes.tag: INSDC first public
attributes.value: 2015-01-09T17:01:37Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T22:15:26Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3135:Kc167_RpL13_IP
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: Kc167
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: genotype
attributes.value: tagged RpL13 expression
attributes.tag: protocol
attributes.value: control conditions with micrococcal nuclease
attributes.tag: sample name
attributes.value: E-MTAB-3135:Kc167_RpL13_IP
attributes.tag: scientific_name
attributes.value: Drosophila melanogaster