N.guentheri Male Torin replicate 4
Total RNA was isolated from pooled brain tissue samples (3 brains per replicate) using MagNA Pure Compact RNA Isolation Kit  on MagNA Pure Compact Instrument (Roshe, USA) according to manufacturer's protocol. The quantity of RNA was assessed using Qubit2.0 Fluorometer (Invitrogen, USA). Quality control was carried out using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The A260/A280 ratios of RNA samples were 1.82.0. The RNA integrity number (RIN) of each sample was not less than 8.0. For cDNA library preparation we used Illumina TruSeq RNA Sample Preparation Kit (Low-Throughput protocol) according to the manufacturer's guidelines. mRNA molecules containing poly(A) tails were isolated from 1 g of total RNA, fragmented and primed for cDNA synthesis in two rounds of purification by the poly-T oligo-attached magnetic beads. The first strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase and random primers. Then cDNA was converted into the double-stranded (ds) cDNA and purified by AMPure XP beads. For creation of blunt ends on the ds cDNA the end-repair reaction was performed. A single A nucleotide was added to the 3 ends to avoid the ligation of blunt ends during the adapter ligation reaction. After adapters ligation with RNA Adapter Indexes, supplied in the kit, the PCR (15 cycles) was performed to amplify the amount of DNA in the library. The concentration of the 21 obtained libraries was evaluated using Qubit2.0 Fluorometer. The quality was checked on Agilent 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent Technologies, USA). cDNA libraries were normalized to 4 nM, pooled together in equal volumes, and sequenced with 75 bp single-end reads on the NextSeq500 System (Illumina, USA).