GSM3960603: P3-E1-069-SN-R; Mus musculus; RNA-Seq
DNA: DNA isolation was performed on frozen mouse midbrain samples using the phenol:chloroform:isoamyl alcohol method. RNA: RNA isolation from frozen mouse midbrain samples was performed using a modified RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). Several changes were made to the standard RNeasy Lipid Tissue Mini kit to improve RNA yield from midbrain samples. First, tissue was homogenized in 200 µL cold Qiazol lysis reagent using a Kimble Kontes pellet pestle (Kimble Chase, Rockwood, TN) for microcentrifuge tubes. Second, after physical homogenization, an additional 800 µL of Qiazol lysis reagent was added to each tissue sample. Third, after initial centrifugation, supernatant was transferred to an Invitrogen Phasemaker tube (ThermoFisher, Waltham, MA) to facilitate transfer of the RNA-containing aqueous layer. As a result of using the Phasemaker tubes, the second centrifugation step was reduced from 15 minutes to 5 minutes. In addition to these changes, the optional DNase digestion step (RNeasy Lipid Tissue Appendix C) was included to improve purity of isolated RNA. DNA (RRBS): RRBS library preparation was performed at the University of Michigan Epigenomics Core. Briefly, 200 ng of genomic DNA was digested for 16-18 hours using MspI, a restriction enzyme that preferentially cuts DNA at CCGG nucleotide sequences. Restriction enzyme-digested DNA was purified using phenol:chloroform extraction and ethanol precipitation in the presence of glycogen and sodium acetate. Precipitated DNA was resuspended in 10 mM Tris-Cl pH 8.5 and processed for end-repair, A-tailing, and ligation of adapters with methylated cytosines. The ligated DNA was further cleaned according to the NuGEN TrueMethyl oxBS module (NuGEN, Redwood City, CA; Cat # 0414-32) instructions with 80% acetonitrile and magnetic beads. Ligated DNA was then eluted in UltraPure water and split into two tubes, one processed for bisulfite conversion only and the other processed for oxidative-bisulfite treatment, according to the TrueMethyl kit's instructions. After bisulfite conversion and cleanup, the samples were amplified using the Roche High Fidelity FastStart system ((Sigma-Aldrich, St. Louis, MO) and TruSeq PCR primers (Illumina Inc., San Diego, CA), according to the following protocol: 94˚C for 5 minutes; 18 cycles of 94˚C for 20 seconds, 65˚C for 30 seconds, and 72˚C for 1 min; 72˚C for 3 minutes. Final library cleanup was done with 1:1 volume of Agencourt AMPure XP SPRI beads (Beckman Coulter, Brea, CA). RNA (RNA-seq): RNA-seq library preparation was performed at the Van Andel Genomics Core. Libraries were prepared from 100 ng of total RNA using the KAPA RNA HyperPrep Kit with RiboseErase (v1.16) (Kapa Biosystems, Wilmington, MA USA). As part of the library prep, RNA was sheared to 300-400 bp. After cDNA synthesis, but prior to PCR amplification, cDNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA).