title: Mutant footprints and total RNA
design: Footprint preparation:  As described [1], the frozen lysate after thawing on ice was supplemented with 5 mM CaCl2. Digestion was carried out at 25ºC in a thermomixer (Eppendorf) shaken at 550 rpm with 750 U micrococcal nuclease (Roche, catalog no. 10107921001) for each 0.5 mg RNA. Digestions were quenched after 1 hr with EGTA to a final concentration of 6 mM. Both digested and control samples were loaded onto 10/45% sucrose gradients (buffered in 10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris pH 8.0, 2 mM DTT). The gradients were separated by ultracentrifugation at 35,000 rpm with SW41 rotor for 2.5 hr at 4ºC. Fractions were harvested from the gradient and monosomes were manually collected by monitoring OD254 nm.  Library preparation:    Ribosome footprints were converted to a cDNA library as previously described [1,2]. Samples were first denatured using SDS to a final concentration of 1% (w/v). Following that, the denatured samples were extracted once with 1 volume of hot acid phenol then once with 1 volume of acid phenol and finally with 1 volume of chloroform–IAA (24:1). The precipitated yield was resuspended in 20 µl of 10 mM Tris pH 7.0. To resolve, 25 mg of RNA was mixed with 2x TBE-urea sample loading buffer (Invitrogen) and loaded on a 15% TBE-urea gel. The gel was run in 1x TBE at 200V then stained for 3 min in SYBR Gold. With the 10 bp ladder (Invitrogen), a band between 28 to 42 bp was excised; gel purified, and resuspended in 10 mM Tris pH 7.0. Dephosphorylate of the 3 ´ ends of the RNA was done by adding T4 PNK (NEB) for 1 hr at 37ºC; followed by heat inactivation. The precipitated RNA was resuspended in 10 µl of 10 mM Tris pH 7.0 . From this, 5 pmol (quantified by Bio-Analyzer) of RNA? was diluted to 5 µl in 10 mM Tris pH 7.0 and ligated for 2.5 hr at 37ºC to 1 µl of 1 µg/µl Linker-1 (5 ´_App/CTGTAGGCACCATCAAT/3 ddC_ 3  ´, IDTDNA) with 8 µl of 50% sterile filtered PEG MW 8000, 2 µl of 100% DMSO (Sigma), 2 µl of 10x T4 RNA ligase buffer (NEB), 1 µl of 40 U/µl RNase Inhibitor (Roche), and 1 µl of T4 ligase 2, truncated (NEB). The ligated products were precipitated and resolved on a 10% TBE-urea gel in 1x TBE at 200V. Using the 10 bp ladder, a  band between 45 and 60 bp was excised. After gel extraction, the products were reverse transcribed using Superscript III (Invitrogen) in a 20 µl reaction volume at 50°C for 30 min [3] with no more than five molar excess of Reverse transcription primer, 5'-(Phos)-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SpC18)-CACTCA-(SpC18)-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGC CTACAG-3'.  NaOH was added to hydrolyze RNA products were (final concentration of 0.1 mM) and incubated at 95°C C for 15 min. A 10% TBE-urea gel was used to resolve the reverse transcribed cDNA. A band between 165 to 180 bp was excised using 10 bp ladder as the standard. After gel purification, the product was resuspended in 15 µl of 10 mM Tris pH 8.0 and circularized with CircLigase (EPICENTRE) at 60°C for 1 hr. To inactivate the enzyme, it was heated at 80°C for 10 min. The circDNA was PCR amplified as previously described [3] with the Forward library PCR primer, 5'-AATGATACGGCGACCACCGAGATCTACAC-3' and an Indexed reverse library PCR primers, 5'-CAAGCAGAAGACGGCATACGAGATNNNNNN GTGACTG GAGTTCA GACGTGTGCTCTTCCG-3' (NNNNNN indicates the reverse complement of the index sequence discovered during sequencing. The twelve forward-strand barcode sequences in supp Table 1). The PCR was done for 7 to 12 cycles using Phusion polymerase (NEB) and the  products were resolved on an 8% polyacrylamide gel in 1x TBE at 180V. Using the 10 bp ladder as the standard, a band between 165 to 180 bp was excised. The purified product was resuspended in 10 µl of 10 mM Tris pH 8.0 and quantified by BioAnalyzer (high sensitivity DNA kit, Agilent).      References          1. Oh E, Becker AH, Sandikci A, Huber D, Chaba R, et al. (2011) Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147: 1295-1308.    2. Ingolia NT (2010) Genome-wide translational profiling by ribosome footprinting. Methods Enzymol 470: 119-142.    3. Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7: 1534-1550.    4. Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147: 789-802.  References        1. Oh E, Becker AH, Sandikci A, Huber D, Chaba R, et al. (2011) Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147: 1295-1308.  2. Ingolia NT (2010) Genome-wide translational profiling by ribosome footprinting. Methods Enzymol 470: 119-142.  3. Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7: 1534-1550.  4. Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147: 789-802.