Polyadenylation and RT of Plasma Cell RNA

TRI Reagent^® (Molecular Research Center Inc., Cincinnati, OH, USA) was used to isolate the total RNA from CD138+ plasma cells. A Qubit™ 2 Fluorometer (Invitrogen™, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) was used to measure the RNA concentration of the extracts. Following that, 200 ng of each total RNA extract were in vitro polyadenylated using E. coli Poly(A) Polymerase (New England Biolabs Ltd., Ontario, ON, Canada) and 80 μM ATP, at 37 °C for 60 min, followed by an inactivation step at 65 °C for 10 min. Subsequently, the in vitro polyadenylated RNA samples were reversely transcribed using MMLV reverse transcriptase (Invitrogen™, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and an oligo-dT–adapter primer, as previously described [56 (link)].