GSM8830175: RNA-seq of excitatory nuclei, replicate 2; Mus musculus; RNA-Seq
The FACS-sorted neuronal nuclei were pelleted and resuspended in 1mL TRIzol. After TRIzol/chloroform phase separation, the aqueous phase was collected in a new 1.5mL tube, an equal volume of 100% ethanol was added. The mixture was loaded onto the RNA Clean&Concentrator-5 columns. After washing once with RNA Wash Buffer, DNase I solution were added into the column and incubated for 15min at room temperature to remove any residual DNA. After washing once with RNA Prep Buffer and twice with RNA Wash Buffer, RNA was eluted from the column with RNase-free H2O. RNA concentrations were quantified with Qubit RNA HS assay kit (Thermo Fisher, cat# Q32852) on Qubit 3.0 Fluorometer (Thermo Fisher, cat# Q33218). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, cat# E7760S) following the manufacturer's instructions. Briefly, rRNAs were depleted from the neuronal nuclei RNA samples using NEBNext rRNA Depletion Kit (NEB, cat# E6350S). The rRNA-depleted RNA samples were fragmented, followed by first strand cDNA synthesis and second strand cDNA synthesis. After purification with Ampure XP beads (Beckman Coulter, cat# A63880), the double-stranded cDNA samples were end repaired, dA-tailed and ligated with adaptors. After purification with Ampure XP beads (Beckman Coulter, cat# A63880), PCR amplification for 12 cycles were performed to enrich adaptor-ligated DNA. The PCR products were purified by Ampure XP beads. The concentrations of the RNA-seq libraries were quantified by Qubit dsDNA HS assay kit (Thermo Fisher, cat# Q32854) on Qubit 3.0 Fluorometer (Thermo Fisher, cat# Q33218) and the peak distributions were measured by Agilent DNA D1000 Screen Tape (Agilent, cat# 5067-5582) on 4150 TapeStation System (Agilent, cat# G2992AA). The indexed RNA-seq libraries were pooled and sequenced on Novaseq 6000 platform with paired end 150bp mode.