Illumina HiSeq 2000 sequencing; Ribosome profiling of Pseudomonas fluorescens SBW25 and its hfq deletion mutant.
Ribosome profiling of Pseudomonas fluorescens SBW25 and its hfq deletion mutant.
SBW25 WT and Δhfq cultures were grown at 28°C in M9 medium supplemented with 0.4% pyruvate and 0.4% casamino acids to the late exponential phase. Cells were harvested by rapid filtration as described in Oh et al. (2011) Cell 147(6):1295-1308. Collected cells were flash frozen in liquid nitrogen and cryogenically pulverized by mixer milling (Retsch). Pulverized cells were thawed and clarified by centrifugation. Resulting lysates were digested with MNase, quenched with EGTA and resolved by sucrose density gradient ultracentrifugation. Ribosome-protected mRNA footprints were processed as previously described in Becker et al.(2013) Nat Protoc 8(11):2212-2239. Sequencing library was prepared as described in Oh et al. (2011) Cell 147(6):1295-1308. Briefly, RNA extracted from monosome fractions was size selected for 28 to 42 nucleotides by running on a 15% TBE-urea gel using a 10 bp ladder as the standard. The 3′ ends of RNA were dephosphorylated by T4 PNK and then ligated to the linker 5′_App/CTGTAGGCACCATCAAT/3 ddC_3′. The ligated products were size selected for 45 to 60 nucleotides and reverse transcribed with no more than five molar excess of 5′_5phosCAAGCAGAAGACGGCATACGAATTGATGGTGCCTACAG_3′. The reverse transcribed cDNA was size selected for 125 to 150 bp and circularised. The circDNA was PCR amplified with 5′_AATGATACGGCGACCACCGA_3′ and 5′_CAAGCAGAAGACGGCATACGA_3′ for 7 to 10 cycles and the amplicons were resolved on an 8% polyacrylamide gel in 1x TBE to excise out a band between 125 to 150 bp. The PCR amplified DNA library was quantified using the Agilent BioAnalyzer kit.