title: Illumina HiSeq 2000 sequencing; A comparison of the effects of duplex-specific nuclease, antisense oligonucleotides, and riboZero rRNA depletion on ribo-Seq data from mouse
design: A comparison of the effects of duplex-specific nuclease, antisense oligonucleotides, and riboZero rRNA depletion on ribo-Seq data from mouse
library_construction_protocol: Murine 17 clone 1 cells were maintained in Dulbecco’s modification of Eagle’s medium supplemented with 10% (vol/vol) fetal calf serum. Cells (107) were plated in a 10 cm dish and upon reaching 100% confluence, cycloheximide (Sigma-Aldrich) was added to 100 µg/ml. After 2 min, cells were rinsed with 5 ml of ice-cold PBS, the dishes submerged in a reservoir of liquid nitrogen for 10 sec, transferred to dry ice and 400 µl of lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide and 25 U/ml TURBO™ DNase (Life Technologies) dripped on. The cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 ºC, the supernatants recovered and stored in liquid nitrogen. For mouse samples, lysates were slowly thawed on ice and 300 µl treated with 7.5 µl RNase 1 followed by incubation for 45 min at room temperature on a rotating wheel. 10 µl of SUPERase-In RNase inhibitor was added, the sample layered onto a 1M sucrose cushion in mammalian polysome buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide) and ultracentrifuged at 28,000 rpm (16 hr, 4 ºC) in a Beckman SW55Ti rotor. Subsequently, all ribosome pellets were resuspended in 200 µl of the corresponding polysome buffer and digested with proteinase K (10 mM Tris-HCl pH 7.5, 10% SDS, 200 µg/ml Proteinase K [New England Biolabs]) for 30 min at 42 ºC. RPFs were recovered by extracting twice with pre-warmed (65 ºC) acidic phenol:chloroform (Life Technologies) and once with chloroform (1:1, vol/vol, buffered with 10 mM Tris pH 7.5, 0.1 mM EDTA), followed by ethanol precipitation. RPFs were resuspended in 10 mM Tris-HCl pH 7.5 and quantified by spectrophotometry. The RNA samples from above were heated at 80 ºC for 2 min, cooled and the 3’ phosphate group removed using T4 polynucleotide kinase (T4 PNK, New England Biolabs) at 37 ºC for 2 hrs in a 20 µl reaction lacking ATP. The RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris-HCl pH 7.5 and ligated in a 20 µl reaction overnight at 14 ºC to a preadenylated 3’-adaptor (5’ rATGGAATTCTCGGGTGCCAAGG 3’) using T4 RNA Ligase 2 truncated K227Q (New England Biolabs). This 3’ adaptor was adenylated using a 5’ DNA adenylation kit (New England Biolabs) following the manufacturer’s instructions. RNA was precipitated, loaded into a 15% (wt/vol) denaturing polyacrylamide gel and ligated RNA fragments migrating between 49 nt and 53 nt excised. The RNA was eluted, precipitated, resuspended as above and 5’ phosphorylated using T4 PNK in the presence of 1mM ATP for 2 hrs at 37 ºC. RNA was concentrated by ethanol precipitation, resuspended in 10mM Tris-HCl pH 7.5 and ligated to a 5’ RNA adaptor (5’ rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC 3’) in a 20 µl reaction overnight at 14 ºC using T4 RNA Ligase (Promega). The fully adapted RNAs were recovered by ethanol precipitation, dissolved in 6 µl 10mM Tris-HCl pH 7.5 and 3 µl of this ligated product annealed to an RT primer (5' GCCTTGGCACCCGAGAATTCCA, 50 pmol) at 65 ºC for 5 min. The RNA was subsequently reverse transcribed at 55 ºC for 50 min in a 20 µl reaction following addition of first strand buffer (Invitrogen, to 1X), 2.5 mM MgCl2, 10 mM DTT, 0.5 µl SUPERase-In and 1 µl SuperScript III (Invitrogen), followed by heat inactivation at 85 ˚C for 5 min. For PCR and ribosomal depletion steps, see referenced manuscript.