GSM4256975: mRNA_R1; Solanum lycopersicum; RNA-Seq
To purify the ribosome-protected fragments (RPFs) samples for cycloheximide  (CHX) datasets, the polysome complexes were isolated from the grounded plant powder with polysome extraction buffer (20 mM Hepes, 100mM KCl, 5 mM MgCl2, recipe from Gao et al. (2015)) containing 100 ug/ml CHX via 13,000g at 40C for 5 min and digested with RNase 1 to purify the RPFs. The purified RPFs were further resolved in 15% TBE-UREA polyacrylamide gel (Invitrogen) and excise the gels slides corresponding to the 26~32 nt region for library construction of CHX-treated samples  as described previously (Hsu et al., 2016). To purify the RNA samples for total RNA datasets, total RNAs was extracted from the aliquot of the polysome extract mentioned above via RNA clean kit (Zymo) and then input to Ribo-Zero rRNA depletion kit (Illumina) for the rRNA removal. To purify the RPFs for LTM datasets, the polysome complexes were isolated from the grounded powder of the LTM-treated plants with polysome extraction buffer via 13,000g at 40C for 5 min. The supernatant were then subjected to purimycin (PUR) treatment based a reported protocol (Gao et al., 2015) and then processed to RPF purification as described above. The purified RNA fragments was used to construct a library for single 75-bp single-end  sequencing following the protocol described previously (Ingolia, N. T., et al., Science, 2009). Ribosome profiling (LTM-seq and CHX-seq) for initiating and elongation ribosomes ; RNA-seq for total mRNA
GEO Accession
GSM4256975