title: GSM8007154: 9852_cort; Mus musculus; RNA-Seq
library_name: GSM8007154
library_construction_protocol: RNA was extracted using the Qiagen RNAeasy kit for brain tissue. A total of 1.3ug of input RNA was used for the contruction of library The raw sequencing reads were aligned to GRCm39 (mm39) using STAR. The number of reads that were aligned to each gene was obtained while mapping using the GENCODE gene annotation. Then we used edgeR to identify differentially expressed genes. For each genotype (“AA” or “SS”) in each tissue (Hippocampus, cortex), we included all samples in a model and compared the two “exposure” groups (RSD vs. No RSD) and then “treatment” groups (minocycline treatment vs. no minocycline treatment) in a pairwise fashion. We also compared across genotype; however, we ensured that only biologically meaningful comparisons were performed. To isolate polyA RNA for library preparation, NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Ipswich, MA) was used with 500 ng good quality total RNA as input. The polyA RNA was enriched using SMARTer Apollo automated NGS library prep system (Takara Bio USA, Mountain View, CA). Next, NEBNext Ultra II Directional RNA Library Prep kit (New England BioLabs) was used for library preparation under PCR cycle number of 9. After library QC and quantification via real-time qPCR (NEBNext Library Quant Kit, New England BioLabs), individually indexed libraries were proportionally pooled and sequenced using NextSeq 550 sequencer (Illumina, San Diego, CA) under the sequencing setting of single read 1x85 bp to generate about 25M reads. Directional polyA RNA-seq was performed by the Genomics, Epigenomics and Sequencing Core at the University of Cincinnati using established protocols as previously mentioned. To summarize, the quality of total RNA was QC analyzed by Bioanalyzer (Agilent, Santa Clara, CA).  After sequencing, fastq files for downstream data analysis were automatically generated via Illumina BaseSpace Sequence.