Instrument: HiSeq X Ten
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: L. rhamnosus ATCC 53103 lysate was prepared following the protocols described previously with minor modifications. Cultures were poured onto chipped ice of the same volume and centrifuged at 4000 × g for 10 min at 4 ℃ for harvest. The resulting cell pellet was washed using 10 ml resuspension buffer [10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris-HCl (pH 8.0) and 1mM chloramphenicol], and centrifuged again at 3000 × g for 5 min at 4 ℃. Cell pellet of each sample was then resolved in 2 ml lysis buffer [20 Mm Tris-HCl (pH 8.0), 100 mM NH4Cl, 10 mM MgCl2, 0.4% Triton X-100, 0.1% NP-40, 100 U/ml of RNase-free DNase Ⅰ (Roche), 0.5 U/µl of SUPERase In RNase inhibitor (Ambion) and 1 mM chloramphenicol]. Resuspended cells were poured over liquid nitrogen in 50 ml tubes, which were prepared with holes on the cap punched by a syringe needle. Then the tubes were placed at -80 ℃ for 30 min for nitrogen evaporation. Frozen cell pellets were pulverized using Freezer/Mill 6770 with the speed of impactor at 10 cycles per second for 2 min in two sets, transferred to a 50 ml tube, and stored at -80 ℃. After nitrogen evaporation, pulverized cells in 50-ml tubes were placed in water bath at 30 ℃ for immediate thaw, after which the cells were kept on ice for 10 min, and then transferred into a 1.5 ml Eppendorf tube. Lysate was aliquoted into 200 µl fractions, and flash frozen by liquid nitrogen and stored at -80 ℃ prior to further use. 50 µl or the cell lysate per sample was used to isolate total mRNAs with Gene JETRNA purification kit (Thermo Fisher Scientific, K0731). The rRNA removal for 5 µg total mRNAs was accomplished using the Ribo-ZeroTM MagneticKit for Gram-negative bacteria (Epicentre, MRZGN126). Afterward, RNAs were purified with RNA Clean & Concentrator kit (Zymo Research, R1015).RPFs from 100 µl lysate per sample were extracted as previously described. Notably, the concentration of Mg2+ in the lysate was supplemented to 15 mM to decrease the dissociation of 70S ribosomes. Clarified lysate was digested using 60 U MNase per one A260 unit of RNA with 5 mM CaCl2 in the reaction system, rotated at 1400 × g for 1 h at 25 ℃ in a thermomixer. The digestion was quenched with 6 mM EGTA. RPFs were isolated with Sephacryl S400 (GE, MicroSpin S-400 HR, 27514001) and purified using Acid Phenol: Chloroform extraction method prior to storage at -80 ℃ for further use. cDNA libraries of total mRNA and RPFs were constructed using the ARTseqTM Ribosome Profiling Kit (Epicentre, RPHMR12126) with different index tag at the reverse primers listed in Table X. Other primers used during the cDNA library construction were same as mentioned in the ARTseq Ribosome Profiling Kit (Epicentre RPHMR12126). Prepared cDNA libraries were tested with Agilent 2100 bio analyzer for quality determination and then subjected to sequencing for quality determination and then subjected to sequencing with 50-bp single end using Illumina.