title: RNA-seq of mus musculus: adult male brain
design: Mice were anesthetized, followed by thoracotomy and intracardiac perfusion with precooled saline. Then, whole brains were isolated and homogenized with TRIzol reagent (ThermoFisher Scientific, #15596026). The integrity of the extracted total RNA was assessed by a Bioanalyzer 2100 (Agilent, CA, USA) and electrophoresis. Poly(A) RNA was purified from total RNA using Dynabeads Oligo (dT) 25 (ThermoFisher Scientific, #61005) and fragmented into small pieces using the Magnesium RNA Fragmentation Module (New England Biolabs, e6150). The cleaved RNA fragments were reverse-transcribed to cDNA by SuperScript III Reverse Transcriptase (ThermoFisher Scientific, #1896649), which was subsequently used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (New England Biolabs, m0209), RNase H (New England Biolabs, m0297) and dUTP Solution (ThermoFisher Scientific, R0133). An A-base was then added to the blunt ends, preparing them for ligation to the indexed adapters. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After heat-labile UDG enzyme (New England Biolabs, m0280) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR. The average insert size for the final cDNA library was 300  50 bp.