For each 1 mg of wet mass, 1 ml Trizol (Invitrogen) was added, and lysis was performed using 0.4 ml of 0.1 mm zirconia/silica beads by three cycles at full speed in a bead beater, including incubation in ice for 30 s between cycles of beating, followed by 1 min of centrifugation at 13,000 rpm, 4°C. Trizol supernatant was transferred to a heavy-lock phase tube (Eppendorf) containing 200 μl chloroform (Sigma), and vigorously shaken for 15 s, during 2 min. After 10 min at room temperature (RT), we centrifuged at 13,000 rpm, RT, for 5 min, and the supernatant (volume ∼540 μl) was transferred to 1.5 ml tubes, to which 270 μl of 2-propanol (Sigma) and 270 μl of 3 M sodium acetate pH 5.2 (Molecular biology grade, Sigma) were added, followed by mixing and incubation overnight at 4°C. Samples were centrifuged at 13,000 rpm, 4°C, 10 min, and the nucleic acids pellet was washed twice with 70% ethanol. After drying and dissolving in RNAse-free water (Sigma), we quantitated nucleic acids using a UV spectrophotometer. We performed genomic DNA digestion in solution using RQ1 DNase (Promega) following the manufacturer's recommendation, and further clean-up was performed using RNeasy kit (Qiagen) according to their recommended protocol. RNA was eluted and quantitated RNA was used to prepare cDNA using Nugen's Ovation RNA-Seq System via single primer isothermal amplification (Catalogue # 7102-A01) and automated on the (BRAVO NGS liquid handler from Agilent). cDNA was quantified on the Nanodrop (Thermo Fisher Scientific). Using Kapa Biosystem's DNA Hyper Plus library preparation kit, (KK8514) cDNA was enzymatically sheared to approximately 150 bp fragments, end repaired and A-tailed. Illumina-compatible adapters with unique indexes (IDT #00989130v2) were ligated on each sample individually. The adapter ligated molecules were cleaned using Kapa pure beads (Kapa Biosciences, KK8002), and amplified with Kapa's HIFI enzyme (KK2502). Each library was then analyzed for fragment size on an Agilent's Tapestation, and quantified by qPCR (KAPA Library Quantification Kit, KK4835) on Thermo Fisher Scientific's Quantstudio 5 before multiplex pooling and sequencing a 2x75 flow cell on the NextSeq500 platform (Illumina) at the ASU's Genomics Core facility.
GEO Accession
GSM4520990