GSM5065743: 2471_cerebellum_male_vehicle; Mus musculus; RNA-Seq
On P10, 24 hours following the final E or V treatment, animals were anesthized using isoflurane vapor, transcardially perfused with phosphate buffered saline containing 5 U/mL heparin, brains were removed, and the hippocampus and cerebellum were microdissected, flash frozen in liquid nitrogen, and stored at -80 degrees Celcius. Hippocampus and cerebellum were homogenized according to manufacturer specification using a Next Advance BBX24B Bullet Blender homogenizer and 0.5 mm glass beads.  RNA was isolated using the Qiagen RNeasy Mini Kit plus on-column DNase Removal Kit. RNA quantity was assessed using the Qubit 3.0 fluorometer with the Qubit Broad-Range RNA Assay Kit and an Agilent Fragment Analyzer with the Standard Sensitivity RNA Gel Kit was used to assess RNA quality.  All samples had an RNA Quality No. ≥ 8.9 (Mean 9.7, Median 10.0).  RNA-seq libraries were prepared using the Illumina TruSeq mRNA Library Prep Kit with TruSeq Unique Dual Indexed adapters and were quantified with the Qubit 1X dsDNA High-Sensitivity NGS Gel Kit.  Libraries were further characterized for functionality with the KAPA Library Quantification Kit and for fragment size using the Agilent Fragment Analyzer with the High-Sensitivity NGS Gel Kit.  Library molarities were calculated followed by dilution and denaturation all according to manufacturer's specification for clustering.  An initial set of n = 5V and 5E were clustered on a cBot and paired-end sequenced on a HiSeq 3000 with a 150-cycle SBS kit for 2X75 reads.  A supplemental set of n = 2V and 2E were clustered on a high-output NextSeq 500 flow cell and paired-end sequenced with a 150-cycle SBS kit for 2X75 reads.