GSM9115524: cont_T6_1; Escherichia coli; OTHER
Total RNA was extracted using the hot phenol method. The aqueous phase was precipitated with 2 volumes of ice-cold ethanol and 1/30 volume of 3 M NaOAc (pH 6.5) for 2 h at -20°C. The pellet was washed with ice-cold 75% ethanol and resuspended in sterilized water. Then the extracted RNA was treated with DNase followed by phenol:chloroform:isoamyl alcohol extraction. The aqueous phase was precipitated again with 2.5 volumes of ice-cold ethanol and 1/30 volume of 3 M NaOAc (pH 6.5) for 2 h at -20°C. The pellet was washed with 75% ethanol and resuspended in sterilized water. cDNA library preparation followed by sequencing was conducted by Filgen, Inc. and Arraystar Inc.. RNA samples were pretreated to eliminate specific modifications that might impede the process (deacylation of 3'–aminoacyl RNA, removal of 3'–cP (2',3'–cyclic phosphate), phosphorylation of 5'–OH, and demethylation of m1A and m3C ). Library preparation was executed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs). The procedural workflow incorporated 3'–adapter ligation, 5'–adapter ligation, cDNA synthesis, PCR amplification, and size selection of PCR products ranging from 134 to 160 bp.