GSM2573140: Autotrophic Ribo rep2; Eubacterium limosum; OTHER
For total RNA extraction, the cells were resuspended in 500 µl of lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Resuspended cells were frozen in liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and the cell debris was removed by centrifugation at 4,000g for 15 min at 4 °C. Total RNA was then isolated using Trizol (Thermo Scientific, Waltham, USA) according to the manufacturer’s instruction. To remove remaining genomic DNA in the purified RNA sample, 4 U of rDNase I (Ambion, Waltham, USA) was treated to the isolated RNA for 1 h at 37 °C, followed by the incubation at 75 °C for 10 min. For ribosome profiling, the CM treated cells were washed using 500 µl polysome buffer that composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 100 µM CM. The cells were resuspended in lysis buffer that composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, 100 µM CM, and 1% Triton X-100. The resuspended cells were frozen in liquid nitrogen and ground with pestle and mortar. The powdered cells were recovered by centrifugation at 4,000g for 15 min at 4 °C, then the supernatant was transferred to a new tube. For further clarification, the supernatant was additionally centrifuged at 16,000g for 10 min at 4 °C. To degrade RNA that unprotected by ribosome, 400 U of MNase (NEB, Ipswich, USA), 2 µl of BSA (1 mg ml-1), 20 µl of 10× MNase buffer were added and incubated at 37 °C for 2 h with gentle rotation. For inactivation of the reaction, 10 µl of EGTA (0.5 M) was added to the sample. Monosome fraction was recovered using Microspin S-400 HR (GE, Pittsburgh, USA) in accordance to the manufacturer’s instruction. The recovered ribosome bound RNA was isolated by using Trizol For RNA-seq, Ribosomal RNAs (rRNA) were removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre, Madison, USA) according to the manufacturer’s instruction, and the rRNA-depleted RNA quality was checked with Agilent 2200 TapeStation system (Agilent Technologies, Santa Clara, USA). Using TrueSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, USA). For ribosome profiling seq, the remaining rRNAs were removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria. For phosphorylation reaction, samples were denatured at 80 °C for 90 s, equilibrated to 37 °C, and incubated at 37 °C for 1 h with 5 µl of 10× T4 PNK buffer (NEB, Ipswich, USA), 20 U of SUPERase In RNase Inhibitor, and 10 U of T4 PNK (NEB, Ipswich, USA). After purification of the RNA samples using RNeasy MinElute Column, the concentration of the purified RNA was measured using Qubit RNA HS assay kit (Invitrogen, Waltham, USA). For library construction, small RNA library prep kit for Illumina (NEB, Ipswich, USA) was used. For differential RNA-seq, the rRNA-depleted RNA was split into three samples for the construction of three different libraries (TEX, TEX, and TAP). For the preparation of TEX library, TerminatorTM 5’-Phosphate-Dependent Exonuclease (Epicentre, Madison, USA) was treated with 2 µl of 10× TerminatorTM Reaction Buffer A (Epicentre, Madison, USA) and 20 U of SUPERase In RNase Inhibitor (Thermo Scientific, Waltham, USA). For the preparation of TEX and TAP libraries, TEX treatment was omitted but 2 µl of 10× TerminatorTM Reaction Buffer A and 20 U of SUPERase In RNase Inhibitor were added. All of the samples were incubated at 30 °C for 1 h, and then the reaction was terminated by adding 1 µl of 100 mM EDTA (pH 8.0). To ligate 5 RNA adaptor, 20 U of RNA 5-polyphosphatase (Epicentre, Madison, USA) was treated to TEX and TEX samples. For TAP sample, the RNA 5-polyphosphatase treatment was omitted. 2 µl of 10× RNA 5’-phosphatase Reaction buffer (Epicentre, Madison, USA) was added to all samples with 20 U of SUPERase In RNase Inhibitor and incubated at 37 °C for 1 h. The samples were purified by phenol-chloroform extraction and ethanol precipitation. 10 pM of 5 RNA adaptor (5’- GUUCAGAGUUCUACAGUCCGACGAUC-3’) was added to the purified mRNA with 2 µl of T4 RNA ligase buffer (Thermo Scientific, Waltham, USA), 2 µl of BSA (1 mg ml-1) (Thermo Scientific, Waltham, USA), 20 U T4 RNA ligases (Thermo Scientific, Waltham, USA), and 20 U of SUPERase In RNase Inhibitor (Epicentre, Madison, USA). The samples were then incubated at 37 °C for 90 min, followed by incubating at 70 °C for 10 min. For cDNA synthesis, 10 pM of the random hexamer 3’ overhanging primer (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN-3’) was added to the purified sample with 1 µl of 10 mM dNTPs (Invitrogen, Waltham, USA) and incubated at 65 °C for 10 min. Subsequently, 2 µl of 10× RT buffer, 2 µl of 100 mM DTT, 4 µl of 25 mM MgCl2, 20 U of SUPERase In RNase Inhibitor (Epicentre, Madison, USA), and 200 U of SuperScript III Reverse Transcriptase (Invitrogen, Waltham, USA) were added. The reaction was incubated at 25 °C for 10 min, at 50 °C for 60 min, 85 °C for 5 min, and cooled down to 4°C. To remove remaining RNA, 2 U of RNase H (Invitrogen, Waltham, USA) was added and incubated at 37 °C for 20 min. The cDNA fraction at a size range between 120 bp and 400 bp was purified on 2% agarose gel using MinElute Gel Extraction kit (Qiagen, Hilden, Germany). The purified library was amplified with indexed primer for Illumina sequencing platform and monitored by CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, USA) with stopping the amplification at the beginning of the saturation point. The amplified cDNA was size-selected at a range between 150 bp and 400 bp, which was then sequenced using the 100 bp read recipe on Illumina Hiseq2500. RNA-seq, Ribosome profiling seq, differential RNA-seq