GSM3045709: DMS-MaPseq on E. coli (Total RNA); Escherichia coli; OTHER
Each bacteria pellet from a 25 ml culture (OD600 ~0.3) was homogeneously resuspended in 200 µl of Buffer A [10 mM Tris pH 8.0; 20% Sucrose; 100 mM NaCl] supplemented with 200 U SUPERase• In™ RNase Inhibitor, by pipetting. 50 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0] supplemented with 1 µl Ready-Lyse™ Lysozyme Solution (Epicentre, cat. R1810M) were added dropwise, and the vial was gently tilted 5 times to ensure homogenous mixing. The sample was then incubated 1 minute at room temperature. 250 µl of Buffer C [0.5% Tween-20: 0.4% NaDOC; 2 M NaCl; 10 mM EDTA] were immediately added dropwise. The sample was then incubated 5 minutes at room temperature. For each 100 µl of lysate, 1 mL of ice-cold TRIzol was added and sample was incubated at 56°C for 5 minutes. RNA was then extracted following manufacturer instructions. 1 μg of DMS-treated RNA, either total, or rRNA-depleted using Ribo-Zero rRNA Removal Kit (Illumina), was fragmented in the presence of 10 mM MgCl2 at 94°C for 8 minutes. RNA was end-repaired by treatment with 1 U rSAP (NEB) at 37°C for 30 minutes. After heat-inactivating the enzyme at 65°C for 5 minutes, RNA fragments were phosphorylated by treatment with 10 U T4 Polynucleotide kinase (NEB) in the presence of 1 mM final ATP at 37°C for 1 hour. After reaction cleanup on RNA Clean & Concentrator™-5 columns (Zymo Research), 3' and 5' adapters were ligated to RNA fragments using the NEBNext® Small RNA Library Prep Set (NEB). RNA was then heat-denatured at 70°C for 5 minutes and incubated at room temperature for 5 minutes. Reverse transcription was carried out in a final volume of 10 μl, in the presence of 1 mM dNTPs, 10 pmol of SR RT primer (NEBNext® Small RNA Library Prep Set kit), 20 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, and 100 U TGIRT™-III Reverse Transcriptase (InGex), by incubating at 50°C for 5 minutes, followed by 2 hours at 57°C. Template RNA was degraded by adding 1 μl of 5 M NaOH and incubating at 95°C for 3 minutes. After reaction cleanup, cDNA was eluted in 20 μl nuclease-free water, and barcodes were introduced by 15 cycles of PCR in the presence of 25 pmol of each primer, and 25 μl NEBNext® High-Fidelity 2X PCR Master Mix. DMS-MaPseq
GEO Accession
GSM3045709