GSM3639498: StrainB-TotalmRNA-Rep1; Mycolicibacterium smegmatis; RNA-Seq
Total RNA were extracted by miRNeasy kit from bacteria lysate. For ribosome footprint library, bacteria suspension was dropped into liquid nitrogen and smashed into powder. The powder was further pulverized with beads beating 4 to 5 times with chilling in liquid nitrogen between each step. After centrifugation at 18500g for 45 min, the cleared lysate was transferred to a new tube supplied with 70U/ml MNase to digested mRNA not protected my ribosome. The pre-digested lysate was laid over a 1M sucrose cushion and centrifuged at 30,000 rpm, 4°C for 20h in Beckman 70Ti rotor. After ultracentrifugation, the ribosome pellet was washed and dissolved in polysome buffer. The dissolved ribosome solution was further digested with MNase at 4°C for 1h. The digested ribosome fraction was incubated with either Flag resin or HA resin at 4°C overnight. After washing of the bound resin, the resin was elued with 3Flag/3HA peptide to isolate the AltRibo, and the remaing flow-through was confirmed as consisting almost entirly of CanRibo. Ribosome footprints were further extracted by miRNeasy Kit. For RNA-seq library, rRNA was romoved by Ribo-Zero rRNA Removal Kit and library was constructed by NEBNext Ultra Directional RNA Library Prep Kit for illumina. For ribosome footprint library, footprints were selected for 24-36nt by marker oligo through 15% TBE-UREA PAGE. The corresponding region was sliced and purified. RNA was further dephosphoylated and ligated with linker. The ligation products were purified again by 15% TBE-UERA PAGE. Reverse transcription was followed up as well as circularization. rRNA was depeleted by biotinylated oligo and the rRNA substracted circularized cDNA template was used for library amplification.
GEO Accession
GSM3639498