characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-01-09T17:01:34Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:18:07Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2421582
characteristics.characteristic: External Id
characteristics.text: DUNDEE
characteristics.characteristic: INSDC center name
characteristics.text: 2015-01-09T17:01:34Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:18:07Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS426770
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2421:Kc167_RNaseI
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 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. Cells were rinsed twice with cold PBS and lysed in 1 ml polysome extraction buffer (1 % deoxycholate, 1 % NP40, 10 mM HEPES (pH 7.4), 350 mM KCl, 5 mM MgCl2, 5 mM CaCl2, 250 uM cycloheximide, 1x protease inhibitors (Sigma, P8340) and 2 ul RiboLock RNase inhibitor (ThermoFisher). Lysates were centrifuged for 10 min at 16000 xg and supernatant (leaving lowest ~25% behind) was used for ribosome profiling.The lysate samples were split in to two different digestion samples and digested with either 200 U of E. coli MBP-RNAse I (New England BioLabs) or 100 U of MN (ThermoFisher) by incubating for 40 min (U2OS) or 60 min (Kc167) at RT with slow mixing. 2 U of DNase I (ThermoFisher) was also added to each U2OS sample at the start of digestion. MN reaction was stopped with 10 mM EGTA. The samples were centrifuged at 6000 xg for 5 min to pellet insoluble material. The ribosomes were separated by sucrose step centrifugation using 0.5 M sucrose in polysome extraction buffer supplied with SUPERase-In (1/500) (Ambion). 0.35 ml lysate was layered on top of 0.2 ml sucrose cushion and centrifuged at 100000 xg for 45 min (55000 rpm using TLA 120.1 rotor). Supernatant was removed and pellet was suspended in 600 ul Qiazol reagent (Qiagen). Samples were incubated for 15 min at 70C with mixing to dissolve the pellet after which 200 ul of chloroform was added, the samples were vortexed and centrifuged for 10 min at 12000 xg at 4C. RNA was precipitated in the presence of Pink Co-precipitant (Bioline) at -80C o/n. RNA was washed and suspended in 20 ul of sterile water and mixed with 80 ul 2x TBE-urea sample buffer (without Xylene Cyanol FF). Samples were run on a 15 % TBE-urea gel, the gel was stained using 1x SYBR Gold (Invitrogen) and 28-35 nt sized RNA was cut out of the gel with the help of oligoribonucleotide size markers (28 and 35 nt RNA oligos). The gel pieces were disrupted and RNA was eluted with 300 mM NaAcetate (pH 5.0), 1 mM EDTA and SUPERase-In for 30 min at 65C. RNA was precipitated, centrifuged and washed with 80% ethanol and then suspended in 5 ul of sterile water. RNA yields were quantified with SYBR Gold using Qubit fluorometer (Invitrogen). All samples had over 1 pmol/ul RNA. 5-15 picomoles of each RNA sample was used for phosphatase and poly-A tailing reaction by mixing 3.25 ul of RNA with the following: 0.5 ul 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), 0.25 ul Yeast poly-A polymerase (600 U/ul, Affymetrix), 0.5 ul 10 mM ATP, 0.3 ul (3 U) T4 polynucleotide kinase (ThermoFisher), 0.2 ul SUPERase-In. Samples were incubated at 37C 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. 4 ul of each sample was mixed with 1 ul 10 uM abasic-oligo dT primers containing random 8 nt sequence used for absolute molecular counting (Casbon et al., 2011; Kivioja et al., 2012), incubated at 72C for 3 min and put on ice. Reverse transcription was started by adding the following to the samples: 5.25 ul H20, 3 ul 5x RT-buffer (ThermoFisher), 0.75 ul 10 mM dNTP, 0.25 ul SUPERase-In and 0.4 ul Maxima Reverse transcriptase (75 U, ThermoFisher). Samples were incubated at 50C for 1 h, heat inactivated at 85C for 6 min after which 0.5 ul RNAse H (ThermoFisher) was added and samples were incubated at 37C for 30 min. Reverse transcription products were purified on 10% TBE-urea gel and cDNA was eluted into 700 ul of DNA gel elution buffer (1x TE + 300 mM NaCl) by incubating o/n at 4C 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 5 ul of circularization mix (0.5 ul CircLigase buffer (10x), 0.25 ul 50 mM MnCl2, 0.25 ul CircLigase II (Epicentre), 1 ul 5 M betaine, 3 ul sterile water). Samples were incubated o/n at 60C and heat-inactivate for 10 min at 80C. cDNA was linearized by adding 3 ul of relinearization supplement (50 mM KCl, 1 mM DTT) followed by 0.7 ul (7 U) APE1 (New England Biolabs). Samples were incubated for 1 hour at 37C. 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: wild type genotype
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
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: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: Kc167_RNaseI
characteristics.characteristic: title