RNA-seq for Micromonas commoda RCC299, mid-exponential, diel
Poly A RNA was isolated from 5ug total RNA (sample received) using Dynabeads mRNA isolation kit (Invitrogen).Isolation procedure was repeated to ensure sample is depleted of rRNA. Purified RNA was then fragmented using RNA Fragmentation Reagents (Ambion) at 70C for 3mins, targeting fragments range 200-300bp.Fragmented RNA is then purified using Ampure XP beads (Agencourt).Reverse transcription was performed using SuperScript II Reverse Transcription (Invitrogen) with an initial annealing of random hexamer (Fermentas) at 65C for 5mins, follow by an incubation of 42C for 50mins and an inactivation step at 70C for 10mins.cDNA was then purified with Ampure XP beads.This was followed by second strand synthesis using dNTP mix where dTTP is replaced by dUTP. Reaction was performed at 16C for 1h.Double stranded cDNA fragments were purified and selected for targeted fragments (200-300bp) using Ampure XP beads.The dscDNA were then blunt-ended, A-tailed, and ligated with library adaptors using Kapa Library Amplification Kit (Kapa Biosystems).Adaptor-ligated DNA was purified using Ampure XP beads.Digestion of dUTP was then performed using AmpErase UNG (Applied Biosystems) to remove second strand cDNA.Digested cDNA was again cleaned up with Ampure XP beads.This is followed by amplification by 10 cycles PCR using Kapa Library Amplification Kit (Kapa Biosystems).The final library was clean up with Ampure XP beads.