description: Protocols: For Ribo-Seq, parental HEK293 and RF1 KO cells were treated with 100 g/ml chloramphenicol and 100 g/mL cycloheximide for 5 min. Cells were rinsed with 5 ml of ice-cold PBS, the dishes were submerged in a reservoir of liquid nitrogen for 10 s and transferred to dry ice. Cells were lysed in 400 l of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 % Triton X-100, 100 g/ml chloramphenicol and cycloheximide). Cells were scraped and lysates clarified by centrifugation for 20 min at 13,000 g at 4C. 200 l aliquots of cell lysates were treated with 7.5 l RNase 1 (100 units/l) at 28C. The reaction was layered onto a 10-30 % sucrose gradient [2ml 10-30% (v/v)] in 50mM Tris-HCl (pH7.2), 20mM Mg(OAc)2, 80mM NH4Cl, 0.1M KCl, 1mM PMSF and centrifuged for 2h 15min at 100,000 gmax at 4C (39,000 rpm, Beckman Coulter TLS-55 rotor) and fractions collected. Subsequently, all fractions containing mitochondrial monosomes, ascertained by western blotting, were pooled and digested with proteinase K (200 g/ml) for 30 min at 42C. RPFs were recovered by extracting twice with pre-warmed (65C) acidic phenol:chloroform and once with chloroform (1:1, v/v, buffered with 10 mM Tris pH 7.5, 0.1 mM EDTA) followed by ethanol precipitation. RPFs (1 g) were separated on 15 % denaturing polyacrylamide gels and RNA species migrating between 25 and 35 nt were harvested. RNA was eluted from the gel slices and ethanol precipitated. The RNA samples from above were heated at 80C for 2 min, cooled and the 3 phosphate group removed using T4 polynucleotide kinase (New England Biolabs) for 2 h at 37C in a 20 l reaction. The RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris-HCl (pH 7.5) and ligated in a 20 l reaction overnight at 14C to a preadenylated 3-adaptor using T4 RNA Ligase 2 (New England Biolabs). RNA was precipitated, loaded into a 15 % denaturing polyacrylamide gel and ligated RNA fragments migrating between 48 and 54 nt were excised. The RNA was eluted, precipitated, resuspended as above, and 5 phosphorylated using T4 PNK in the presence of 1 mM ATP for 2 h at 37C. RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris-HCl (pH 7.5) and ligated to a 5 RNA adaptor overnight at 14C using T4 RNA Ligase (New England Biolabs). The fully adapted RNAs were recovered by ethanol precipitation, dissolved in 6 l 10 mM Tris-HCl (pH 7.5), and ligated product annealed to an RT primer for 5 min at 65C. The RNA was subsequently reverse transcribed for 50 min at 55C in using SuperScript III according to manufacturers instructions, followed by heat inactivation for 5 min at 85C. Standard PCR reactions were used to prepare amplicons using forward primer RP1 and RPIX as reverse primer, where X is primer number (1-24). PCR reactions were loaded onto 10 % non-denaturing polyacrylamide-TBE gels and products of ~150 bp were excised from the gel and eluted. These amplicon libraries were ethanol precipitated and resuspended in 15 l 10 mM Tris-HCl (pH 7.5). Ribosomal RNA was depleted from Ribo-seq samples at the library amplicon stage; 12 L of the relevant Ribo-seq library was mixed with 4 L of 4 hybridization buffer (200 mM HEPES pH 7.5 and 2 M NaCl) and denatured at 98C for 2 min. DNA was re-annealed for 5 h at 68C prior to addition of 2 L of 10 DSN master buffer and 2 L of DSN (4 units, Evrogen). Digestion was allowed to proceed for 25 min at 68C, before addition of 20 L 10 mM EDTA and incubation for a further 5 min at 68C. DNA was recovered by a single extraction with phenolchloroform (1:1, vol/vol) followed by ethanol precipitation and resuspended in 4 L 10 mM TrisHCl pH 7.5. The treated amplicon library was subjected to another round of PCR (as above) and the resulting library subjected to a second round of DSN treatment.
attributes.tag: ENA first public
attributes.value: 2022-05-14
attributes.tag: ENA last update
attributes.value: 2022-05-14
attributes.tag: External Id
attributes.value: SAMEA14321844
attributes.tag: INSDC center alias
attributes.value: KAROLINSKA INSTITUTE
attributes.tag: INSDC center name
attributes.value: KAROLINSKA INSTITUTE
attributes.tag: INSDC first public
attributes.value: 2022-05-14T00:11:56Z
attributes.tag: INSDC last update
attributes.value: 2022-05-14T00:11:56Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11687:Sample2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HEK293
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo stage
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: RK1 knockout
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-11687:Sample2