GSM4435451: VC_WT_log_2; Vibrio cholerae; ncRNA-Seq
Bacterial cells in a culture medium or cecal fluid were harvested by centrifugation. Total RNA was immediately extracted from cell pellets with Trizol according to the manufacture's instruction. The tRNA fraction was cut out from 10% TBE-Urea gels and recovered by isopropanol precipitation. tRNA fraction (250 ng) was deacylated in 500 ul of 100 mM Tris-HCl pH 9.0 at 37 °C for 1 hr and recovered by isopropanol precipitation. After dephosphorylation with alkaline phosphatase from calf intestine (New England Biolabs), tRNAs were ligated to 100 pmol of 5' adenylated and 3' end-blocked DNA oligo (3' linker) using truncated T4 RNA ligase at 25 °C for 2.5 hr in 25% PEG 8000. The ligated product was purified on a 10 % TBE-Urea polyacrylamide gel (Thermo Fisher Scientific) as above. Half of the recovered ligated tRNAs were reverse transcribed with 5 pmol TGIRT-III (InGex) in 100 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 450 mM NaCl, 5 mM MgCl2, 5 mM DTT, 1 mM dNTPs, and 1.25 pmol primer (ocj485) at 60 °C for 1 hr. After the elimination of template RNAs by alkali treatment, cDNA was purified on a 10 % TBE-Urea polyacrylamide gel. The single stranded cDNA was then circularized using 50 U of CircLigase II(Epicenter) at 60 °C for 1 hr, followed by addition of another 50 U of CircLigase II for an additional 1 hr at 60 °C. cDNA was amplified using Phusion DNA polymerase (New England Biolabs) with o231 primer and index primers. After 12-18 rounds of PCR amplification, the product was gel purified from an 8 % TBE-Urea polyacrylamide gel (Thermo Fisher Scientific).
GEO Accession
GSM4435451