GSM8620134: OMA-1 mutant 4-cell, Total RNA-seq, batch12, rep3, read1; Caenorhabditis elegans; RNA-Seq
We used a slightly modified version of the previously described Ribo-ITP protocol 3 and a detailed protocol can be obtained from (https://ceniklab.github.io/ribo_itp/) which can be referred to for more details. In brief, the PCR tubes with 9 embryos were centrifuged to collect them at the bottom. We then added 2.5µl of 100µg/ml cycloheximide to the PCR tubes and we lysed the embryo by freeze thawing them in liquid nitrogen. We then added 2.5 µl of 2x lysis buffer (40mM Bistris, 2% Triton-X-100, 10mM MgCl2, 10 mM CaCl2) and mixed well by pipetting. To obtain ribosome protected fragments, we added 1µl of 1:50 diluted MNase and incubated at 37°C for 30 mins. The digestion was then stopped using 1µl EGTA and vortexed for at least 30 seconds and stored on ice. The microfluidics chip for ribosome profiling via isotachophoresis was setup and run as provided in 3. The ribosome protect fragments were collected in Dephosphorylation Buffer from the Diagenode. RNA libraries were prepared using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB #E6420S/L), following the manufacturer's protocol with some modifications. Briefly, embryo samples, each consisting of 9 embryos, were collected in PCR tubes containing 4.5 µl of water. These samples underwent a freeze-thaw cycle using liquid nitrogen to lyse the cells and release the RNA. For library preparation, we employed a The library preparation was conducted as described in the D-Plex Small RNA-seq Kit ( Diagenode Cat. No C05030001). We made a few modifications to the kit's protocol, and they were as follows. At the RNA-tailing step we used 0.5µl of Dephosphorylation Reagent along with 0.5µl of T4 Polynucleotide Kinase (New England Biolabs M0201L). Upon adding these two reagents the mixture is incubated for 25 mins at 37°C. Next, during the reverse transcription with template switching, we diluted the STSO by half and used 2µl in the mixture. We used 16 cycles for the final PCR amplification to produce the DNA library. RNA libraries were prepared using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB #E6420S/L), following the manufacturer's protocol with some modifications. Briefly, embryo samples, each consisting of 9 embryos, were collected in PCR tubes containing 4.5 µl of water. These samples underwent a freeze-thaw cycle using liquid nitrogen to lyse the cells and release the RNA. For library preparation, we employed a two-step PCR amplification process. The initial cDNA enrichment PCR was performed for 14 cycles, followed by a final library amplification PCR for 8 cycles.