GSM2873982: 1511_Schizo_Olig2; Homo sapiens; RNA-Seq
nuclear extraction: Tissue isolated from Brodmann Area 46 from 40 individuals with schizophrenia and 49 matched controls.700 mg of frozen postmortem brain was homogenized with lysis buffer using a dounce homogenizer. Brain lysate was placed on a sucrose solution to create a concentration gradient. Ultracentrifuge was used to separte nuclei and cytoplasmic fraction. FANS protocol: Nuclei were resuspended in PBS plus RNAse inhibitors and incubated with mouse alexa488 conjugated anti-NeuN (1:200) (#MAB377X, Millipore, Billerica, MA) and rabbit alexa555 conjugated anti-OLIG2 (1:75) (#AB9610-AF555, Millipore) antibodies with 0.5% BSA. Immuno-labeled nuclei were collected as NeuN-positive or OLIG2-positive populations by fluorescence-activated cell sorting (FACS). RNA extraction: Total RNA were purified from each nuclei population using a ZR-Duet DNA/RNA MiniPrep (Plus) kit according to the manufacturer's instruction. 200 ng total RNA from each sample was treated for ribosomal RNA (rRNA) removal using the Low Input RiboMinus Eukaryote System v2 (#A15027, ThermoFisher) according to the manufacturer's instruction. Total RNAs with an average RIN value of 7.5±0.16 were used for RNA-seq library preparation. 50 ng of total RNA after rRNA removal was subjected to fragmentation, first and second strand syntheses, and clean up by EpiNext beads (#P1063, EpiGentek, Farmingdale, NY). Second strand cDNA was adenylated, ligated and cleaned up twice by EpiNext beads. cDNA libraries were amplified by PCR, and cleaned up twice by EpiNext beads. cDNA library quality was quantified by a 2100 Bioanalyzer using an Agilent High Sensitivity DNA Kit (#5067-4626, Agilent, Santa Clara, CA). Barcoded libraries were pooled and underwent 75 bp single-end sequencing on an Illumina NextSeq 500.