GSM5643090: NusA-NusG- sample 3 [dAdG-3]; Bacillus subtilis; OTHER
Cell suspension was digested with lysozyme and RnaseI, lysate was layered on a sucrose cushion, nucleoid with RNAP and nascent RNA pelleted by centrifugation, digested with DNases, native transcription elongation complexes purified by immobilization with Ni-NTA agarose, nucleic acids were isolated and purified, RNA extracted. Purified nascent RNA was ligated to a 5′-adenylated barcode DNA linker, recovered by phenol extraction/ isopropanol precipitation, reverse-transcribed, and digested with RNase H. The resulting cDNA was fractionated through a urea-polyacrylamide gel, followed by staining with SyBR Gold. Bands of the correct length were excised from the gel, DNA was extracted with water and precipitated with isopropanol. The resulting single-stranded cDNA was circularized with CircLigase and used as a template for PCR amplification with a pair of adapters that contained Illumina i5 and i7 barcodes. The resulting sequencing libraries were purified by electrophoresis in native polyacrylamide gels. SYBR gold-stained gels were imaged under ultraviolet light, and slices containing PCR products of interest were excised. DNA was extracted with water and precipitated with isopropanol. Library recovery was estimated by electrophoresis of DNA samples followed by SYBR gold staining and quantification using reference DNA samples and ImageQuant software. The quality and concentration of DNA libraries was also determined using a Bioanalyzer DNA high-sensitivity chip and qPCR.