Total RNA was extracted from GAS grown in THY to early exponential phase with the FastPrep FP 120 kit (Qbiogene, Carlsbad, CA). RNAprotect (Qiagen, Valencia, CA) treated GAS were centrifuged and resuspended in 300 ul 5 mM ammonium aurintricarboxylate before transferring to FastPrep Blue tubes containing 450 ul CRSR-Blue(Qbiogene), and 450 ul acid phenol:chloroform (5:1, pH 4.5; Ambion, Austin, TX). The mixture was homogenized (11 sec at speed #4), heated at 65 oC for 15 min, and centrifuged at 16,000xg for 10 min. The aqueous phase was collected and concentrated to 100 ul with a vacuum concentrator (Eppendorf, Germany). The concentrate was purified using the Qiagen RNeasy 96 kit following the manufacturer's protocol (Qiagen, Valencia, CA).
Label
biotin
Label protocol
Approximately 4 µg of cDNA was processed to produce biotinylated cDNA targets.
