GSM2052448: RNaseE_HTF_2; Escherichia coli O157:H7 str. Sakai; OTHER
Cells were treated with 1800mJ of UV-C and lyzed by vortexing with zirconia beads in lysis buffer. RNasE-RNA complexes were purified over M2 anti-FLAG resin, trimmed with RNase A/T1 and repurified under denaturing conditions using Ni-NTA resin. RNA 5' and 3' end were prepared for ligation by decapping 5' PPP with TAP, phosphorylation of 5'OH with 32P-ATP and PNK, and dephosphyorlation of 3'P with TSAP. RNA linkers were added using T4 RNA ligase. RNaseE-RNA complexes were eluted and seperated by SDS-PAGE, transferred to a nitrocelluose membrane and extracted. Protein was removed by Proteinase K digestion and RNAs purified by phenol:chloroform extraction. Extracted RNAs were converted to cDNA using Superscript III and PCR amplified. PCR products between ~150 and 250 bp were gel extracted and submitted for Illumina sequencing.
GEO Accession
GSM2052448