GSM2462938: mRNA-seq 37°C in WT with plasmid expressing mini-ORF CUG; Escherichia coli; RNA-Seq
Extraction was performed as described previously (Li et al., 2012; Oh et al., 2011). For ribosome profiling, 200 mL of cell culture were filtered rapidly and the resulting cell pellet was flash-frozen in liquid nitrogen and combined with 650 µL of frozen lysis buffer (10 mM MgCl2, 100mM NH4Cl, 20mM Tris-HCl pH 8.0, 0.1% Nonidet P40, 0.4% Triton X-100, 100 U/mL DNase I (Roche), 1mM chloramphenicol). Cells were pulverized in 10mL canisters pre-chilled in liquid nitrogen. Lysate containing 0.5 mg of RNA was digested for 1 h with 750 U of micrococcal nuclease (Roche) at 25°C. The ribosome-protected RNA fragments were isolated using a sucrose gradient followed by hot acid phenol extraction. For mRNA-seq and DMS-seq, cells were pelleted by centrifuging for 2 min at 8000rpm. Total RNA was then hot acid phenol extracted. Ribosomal RNA and small RNA were removed with MICROBExpress (Ambion) or Ribozero (Epicenter) and MEGAclear (Ambion), respectively. The ribosome footprints and fragmented mRNA were ligated to miRNA cloning linker-1 (IDT) using truncated T4 RNA ligase 2 K227Q. The ligated RNA fragments were reverse transribed using the primer 5Phos/GATCGTCGGACTGTAGAACTCTGAACCTGTCGGTGGTCGCC GTATCATT/iSp18/CACTCA/iSp18/CAAGCAGAAGACGGCATACGAATTGATG GTGCCTACAG. The resulting cDNA was circularized with CircLigase (Epicentre) and PCR amplification was done as described previously (Ingolia et al., 2009).
GEO Accession
GSM2462938