# characteristics.description[].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. 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, 250uM cycloheximide, 1x protease inhibitors (Sigma, P8340) and 2ul 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 600ul Qiazol reagent (Qiagen). Samples were incubated for 15 min at 70 C with mixing to dissolve the pellet after which 200ul 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 20ul of sterile water and mixed with 80ul 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 65 C. RNA was precipitated, centrifuged and washed with 80% ethanol and then suspended in 5ul of sterile water. RNA yields were quantified with SYBR Gold using Qubit fluorometer (Invitrogen). All samples had over 1 pmol/ul RNA. For comparisons of digestion conditions U2OS and HeLa cells were treated with FBS and cycloheximide as before and lysed with 0.7 ml polysome extraction buffer. The lysis buffer was modified for each sample: Control samples (buffer as before), low NaCl sample (100 mM NaCl, no KCl), low KCl sample (100 mM KCl), TritonX-100 sample (0.5 % TritonX-100, no deoxycholate or NP40), high MN sample (6x more MN, with 2.5h incubation instead of 40 min, buffer as before). In addition a sample without FBS pre-treatment was made using control polysome extraction buffer. After lysis samples were processed as before, with the exception of high MN samples, which was digested with 600 U of MN for 2.5 h followed by 20 mM EGTA treatment. 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).