GSM3109309: MS2-MicF; Escherichia coli K-12; OTHER
Cells were resuspended in 3 mL of buffer A and lysed using a French Press (430 psi, four times). Lysate was then cleared by centrifugation. The soluble fraction was subjected to affinity chromatography. The column was prepared by adding 100 µL of amylose resin (New England Biolabs) to Bio-Spin disposable chromatography columns (Bio-Rad). The column was washed with 3 mL of buffer A. Next, 200 pmol of MS2-MPB protein was immobilized on the amylose resin, and the column was washed with 2 mL of buffer A. The cleared lysate was then loaded onto the column, which was washed with 8 mL of buffer A. RNA was eluted from the column with 1 mL of buffer A containing 15 mM maltose. Eluted RNA was extracted with phenol-chloroform, followed by ethanol (3 vol) precipitation of the aqueous phase in the presence of 20 mg of glycogen. RNA samples were treated with TURBO DNA-free (DNase treatment) and precipitated again. cDNA libraries were prepared with ScriptSeq™ RNA-Seq Library Preparation Kit (Illumina)
GEO Accession
GSM3109309