GSM8497210: Monosomes, heat shock, Rep3; Leishmania major; RNA-Seq
RNA isolation was made using Direct-zol TM RNA MiniPrep (Zymo Research, Cat: R2052) following the manufacturer's instructions. The RNA was recovered in 30 µL nuclease-free water and quantified by NanoDrop™ One/OneC. Paramagnetic beads coupled with oligo d(T)25 were combined with total RNA to isolate poly(A)+ transcripts based on NEBNext® Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs Inc., Massachusetts, USA). Prior to first strand synthesis, samples are randomly primed (5´ d(N6) 3´ [N=A,C,G,T]) and fragmented based on manufacturer's recommendations. The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 40 minutes at 42⁰C. All remaining steps for library construction were used according to the NEBNext® Ultra™ II Non-Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc., Massachusetts, USA). Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation D1000 ScreenTape (Agilent Technologies Inc., California, USA). Final library size was about 430bp with an insert size of about 300bp. Illumina® 8-nt dual-indices were used.