title: Illumina HiSeq 2000 sequencing; Ribosome footprinting upon oxidative stress treatment for wild-type and catalase2 knock-out mutants
design: Ribosome footprinting upon oxidative stress treatment for wild-type and catalase2 knock-out mutants
library_name: CAT3
library_construction_protocol: Middle aged leaves of 15 individual plants per line were sampled, pooled and frozen in liquid nitrogen. Additionally, the same number of leaves of plants grown in moderate light and elevated CO2 was harvested and served as controls. This entire setup was repeated twice to obtain three biological repeats of each time-point analysed per genotype.  Catalase knock-out (cat2-2) and wild-type (WT) Arabidopsis thaliana plants, ecotype Columbia-0 (Col-0), were grown under elevated CO2 concentrations (3,000 ppm) to impair photorespiration. Individual plants were organized according to a completely randomized block design in a controlled climate chamber (Votsch Industrietechnik) in a 12 h day/12 h night regime (relative humidity of 50%, 21°C and an irradiance of 100 μmol m-2 s-1).  For high light (HL) treatments, 5 week-old plants were transferred to a Sanyo Fitotron plant growth chamber at dawn in ambient air conditions (400 ppm CO2, relative humidity of 55%, 21°C) and continuous HL irradiation of 1,200 μmol m-2 s-1 for 1, 2 or 3 hours.  600 μl supernatant was used for the isolation of ribosome-protected footprints.  Digestion of non-protected mRNA was done by adding 1000 U of RNaseI (Thermo Fisher Scientific) followed by incubation for 55 min with gentle agitation (400 rpm) at 25°C (Thermoblock). This reaction was stopped by adding 600 U of SUPERase•InTM RNase Inhibitor (Thermo Fisher Scientific) and the reaction mixture loaded on top of a 1 M sucrose cushion in 20 mM HEPES-KOH pH 7.4, 5 mM MgCl2, 100 mM KCl, 2 mM DTT, 100 μg/ml CHX and 100 U/ml Superase.In. Ultracentrifugation was performed in a TLA-120.2 rotor at 75,000 rpm for 4 h at 4°C. The supernatant was removed and the RNA pellet was solubilized in 570 μl of 10 mM Tris HCl (pH 7) and 30 μl of 20% SDS. Samples were heated to 65°C and ribosome footprints were purified using the SDS acid phenol:chloroform:isoamyl alcohol (125:24:1) procedure.  From the harvested leaf material used for proteome analysis, 2 ml was crushed in liquid nitrogen to which 2 ml of lysis buffer (10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100 mM KCl, 1%Triton X-100 with freshly added 2 mM DDT, 100 μg/ml cycloheximide (CHX) and 1 x Complete EDTA-free protease inhibitor cocktail (Roche)) was added. Samples were put on ice with frequent mixing and then filtered over paper spin cups (Pierce) by a 1 min centrifugation (4°C) at 100 x g followed by a 5 min centrifugation at 1,000 x g. Supernatants were transferred to new Eppendorf tubes, centrifuged for 15 min at 16,100 x g (4°C) and incubated on ice for 10 min. Another centrifugation step was performed at 16,100 x g and the supernatant was transferred to Eppendorf tubes.  Footprints were loaded on a 15% TBE-urea denaturing PAGE gel (Thermo Fisher Scientific) and run for 65 min at 200 V for subsequent size selection (28-34 nt), after which the extracted gel fragments were dephosphorylated with 10 U of T4 polynucleotide kinase (New England Biolabs) and rRNA depleted using the Ribo-ZeroTM Magnetic Kit (Plant Leaf, Epicentre). This was followed by ligation to a universal cloning linker (New England Biolabs), reverse transcription with SuperScript III (Thermo Fisher Scientific), circularization by CircLigase (Illumina) and PCR amplification using Phusion polymerase (New England Biolabs).
attributes.tag: Experimental Factor: catalase 2 KO  (cat2-2)
attributes.value: genetic modification
attributes.tag: Experimental Factor: high light
attributes.value: stimulus
attributes.tag: Experimental Factor: 3
attributes.value: time