GSM7780049
RNA was isolated and purified using RNEasy Plus Mini Kit (QIAGEN, Hilden, Germany). Paramagnetic beads coupled with oligo d(T)25 are combined with total RNA to isolate poly(A)+ transcripts based on NEBNextR Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs Inc., Massachusetts, USA). Prior to first strand synthesis, samples are randomly primed (5L d(N6) 3L [N=A,C,G,T]) and fragmented based on manufacturerfs recommendations. The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42.C. All remaining steps for library construction were used according to the NEBNextR Ultra. II Directional RNA Library Prep Kit for IlluminaR (New England BioLabs Inc., Massachusetts, USA). Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., California, USA). Final library size was about 500bp with an insert size of about 350bp.