GSM6886871: RNA-seq, LRPPRC KO + LSFC point mutant, rep 2; Homo sapiens; RNA-Seq
For mitoribosome profiling, growth medium was aspirated and cells were quickly rinsed once with ice-cold 1X PBS ph:7.4. Lysis was performed on ice in a glass homogenizer with a loose setting. We used 50 ml of lysis buffer for 106 cells. We used lauryl maltoside at 0.25% in combination with 50 mM NH4Cl, 20 mM MgCl2, 0.5 mM DTT, 10 mM Tris pH: 7.5 and 1X EDTA-free Protease inhibitor cocktail (Roche). For Cytoribosome profiling, cell cultures growth and lysis were performed as for mitoribosome profiling. We used lysis and buffer conditions described in Shalgi et al, 2013, for polysome profiling. For RNA-seq, some of the lysates prepared for mitoribosome profiling were reserved and  further treated with 90 ug/mL of Proteinase K and 0.5 % SDS at 42 0C for 20 minutes. All libraries were prepared as described in Ingolia et al, 2012, with a few modifications. For mitoribosome profiling we did not rRNA deplete. For RNA-seq we included gel purified RNA sizes of 30-70 nucleotides. For all libraries we introduced randomized nucleotides to serve as a unique molecular identifier (UMI) to the linker ligation at 3' (/5rApp/NNNNNNNNNNCTGTAGGCACCATCAAT/3ddC/) and the reverse transcription step at 5'(/5Phos/NNNNGATCGTCGGACTGTAGAACTCTGAACCTGTC/iSp18/CACTCA/iSp18/CAAGCAGAAGACGGCATACGAGATATTGATGGTGCCTACAG).