RNA-CASing. RNA-CASing

The specificity of the RNA-CASing process was analysed by Next-Generation Sequencing. Therfor small RNAs were isolated from purified proteins of Escherichia coli and subjected to Illumina sequencing or nanopore sequencing. Overall design: cDNA libraries were created with the NEBNext® Multiplex Small RNA Library Pret Set for Illumunia according to the manufacturer’s instructions. 100 ng of input RNA was used and amplified cDNA libraries were separated by native PAGE for size selection of 120-250 bp. E. coli small RNA isolates were then subjected to Illumina. Extracted RNA was treated with E. coli Poly(A) Polymerase (NEB) for 30 min 37°C to add a poly(A) tail and then processed for Nanopore sequencing with the Direct RNA Sequencing Kit according to the protocol provided by Oxford Nanopore Technologies.