GSM8214650: Neuro2a Passage 6 Experimental 3; Mus musculus; RNA-Seq
Total RNA was extracted using the MagMax mirVana Total RNA Isolation Kit (Catalog Number A27828, Thermo Fisher Scientific, USA). Samples were processed using an acid phenol-chloroform extraction protocol and the RNA was eluted in molecular-grade water. The RNA was treated with DNase I (15 min at 25°C) and purified using the RNA Clean & Concentrator-5 kit (Catalog Number R1014, Zymo Research, USA), as per the manufacturer's instructions. Purified RNA was eluted in molecular-grade water and the purity and concentration of the samples were evaluated on a NanoDrop 1000 instrument. Library preparation and subsequent sequencing was performed by LC Sciences (Houston, TX, USA). The total RNA quantity and purity were analyzed on a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511). High-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.  mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations at elevated temperatures (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) at 94°C 5-7min). The cleaved RNA fragments were then reverse-transcribed to create the cDNA using the SuperScriptTM II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesize U-labeled second-stranded DNAs using E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95°C for 3 min; 8 cycles of denaturation at 98°C for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 30 sec; and then final extension at 72°C for 5 min