GSM7430309: Ribo_Harringtonin_120s_1; Homo sapiens; OTHER
HEK cells  were cultured in a 10 cm dish in preparation for the RocA treatment, and in a 6-well plate for the heat shock treatment. The cells were then lysed in 500 µl and 100 µl of lysis buffer. Human skin cells cultured in a well of 6-well plates were lysed in 100 μl of lysis buffer. The mouse forebrain was dissected on ice and disrupted using a loose fitting Dounce homogenizer in 3 mL ice-cold homogenizing buffer.The homogenate was treated with an equal volume of 2× lysis buffer and incubated for 10 min on ice. S2 cells, cultured for 3 days, were seeded in 10 cm dishes. To these cells, 10 μl of 100 mg/ml cycloheximide was added, followed by immediate centrifugation at 1,000 × g to collect the cell pellet. The pellet was then washed with PBS and treated with 400 μl of lysis buffer. E. coli MG1655 cells grown overnight at 37°C in LB media were diluted 1:100 in fresh LB media and grown at 37°C until the OD600 was 0.4-0.5. Cells were then collected by filtration and snap frozen in liquid nitrogen. The frozen cell pellet and lysis buffer were disrupted using a multi-beads shocker. For yeast, cells collected in ribosome profiling lysis buffer were pulverised using a Multi-Beads Shocker with a pre-cooled chamber and ball using liquid nitrogen. 0.2 g of frozen Arabidopsis wild-type (Col-0) seedlings were combined with 400 μl of Arabidopsis lysis buffer.This mixture was then subjected to grinding using a multi-bead shocker For ribosome profiling, RNase I treatment, ribosome pelleting, size selection, preadenylated linker ligation, rRNA depeletion, reverse transcription, cDNA circularization, and PCR amplification..For RNA-Seq, TruSeq Stranded mRNA Library Prep Kit (Illumina) or SMARTerseq (Illumina). For Escherichia coli, MNase footprinting, ribosome pelleting, RNA extraction, preadenylated linker ligation, rRNA depeletion, reverse transcription, cDNA circularization, PCR amplification (for ribosome profiling)