Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Total RNA was isolated using Ribozol (Amresco) according to the manufacturer’s manual and further purified using the RNeasy Plus Mini Kit (Qiagen) for removal of gDNA. Residual gDNA was digested with TURBO DNA-freeTM DNaseI (Ambion) and the RNA was quantified by NanoDrop. Isolated total RNA was PolyA-selected using the Oligotex mRNA Mini Kit (Qiagen, #70022) according to manufacturer’s instructions. Subsequently, 2µ of PolyA-selected total RNA equivalent were fragmented by alkaline hydrolysis (5x fragmentation buffer: 200mM Tris acetate pH 8.2, 500mM potassium acetate, 150mM magnesium acetate) for 1min 45sec at 85°C in a 150µl volume and precipitated as previously described in (Hoeijmakers, Bártfai and Stunnenberg 2013), followed by two TURBO DNase treatments (Ambion, #AM2238) to remove remnants of genomic DNA . Further library preparation for strand-specific RNA-seq was carried out as in (Kensche et al. 2015). In short, first strand cDNA synthesis was performed with AT-corrected Random N9 primers (76% AT) and in presence of 0.2µg Actinomycin D (Thermo 446 Fisher Scientific) to prevent unwanted DNA-dependent second strand cDNA synthesis. To maintain the strand-specific information during second strand synthesis dTTPs were replaced with dUTPs. Resulting double stranded cDNAs (1.9-3.2ng) were end repaired and extended with 3’ A-overhangs. After ligation of NextFlex adapters (Bio Scientific, #514122), libraries were treated with USER enzyme (NEB, #M5505L) to specifically degrade the dUTP-containing second strand and subsequently amplified by PCR (98°C for 2min; 4 cycles of 98°C for 20sec, 62°C for 3min; 62°C for 5 min) using KAPA HiFi HotStart ready mix (KAPA Biosystems, #KM2602) and NEXTflex primer mix (Bio Scientific, #514122). Next, libraries were size-selected for 300-400 bp fragments using 2% E-Gel Size Select agarose gels (Invitrogen, #G6610-02) and amplified for another 12 cycles using the same PCR protocol as above. Subsequent depletion of adapter dimers and library purification was performed using Agencourt AMPure XP beads (Beckman Coulter, #A63880) in a 1:1 library beads ratio.