GSM7068694: A909 dCas9_covRPS OD1_2 biol rep2; Streptococcus agalactiae CNCTC 10/84; RNA-Seq
ThermoFisher MagMAX Viral/Pathogen Ultra Nucleic Acid Isolation reagents (cat. #A42356) on a Hamilton Nimbus/ThermoFisher Kingfisher Presto integrated robotic molecular purification instrument Strand-specific, dual unique indexed libraries for sequencing on all Illumina platforms were made using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Manufacturer protocol was modified by diluting adapter 1:30 and using 3 μl of this dilution. Size selection of the library was performed with AMPure SPRI-select beads (Beckman Coulter Genomics, Danvers, MA). Glycosylase digestion of adapter and 2nd strand was done in the same reaction as the final amplification to avoid further cleanups. Sample input for this method was ribosomal RNA reduced RNA. Libraries were assessed for concentration and fragment size using the DNA High Sensitivity Assay on the LabChip GX Touch (Perkin Elmer, Waltham, MA). The library concentrations were also assessed by qPCR using the KAPA Library Quantification Kit (Complete, Universal; Kapa Biosystems, Woburn, MA).