GSM1880814: P2 fraction, EE5 replicate; Mus musculus; RNA-Seq
On day 14 of the behavioral sensitization paradigm, mice were sacrificed by cervical dislocation 4 hours following i.p. injection. Immediately afterward, brains were removed and chilled for one minute in ice-cold 1x phosphate buffered saline. The frontal pole was dissected by making a cut rostral of the optic chiasm and then removing the olfactory bulbs. Excised tissue was stored in a tube on ice for less than 8 minutes before processing for synaptoneurosome isolation. Fresh tissue from 4 animals was pooled (approximately 0.45 g) and manually homogenized utilizing a 15 ml Potter-Elvehjem Safe-Grind® tissue grinder (#358009, Wheaton, Millville, NJ) and diluted 1:10 in synaptoneurosome homogenization buffer. The buffer consisted of 0.35 M nuclease free sucrose (CAS #57-50-1, Acros Organics, NJ), 10 mM HEPES (#15630-056, Life Technologies, Carlsbad, CA), and 1 mM EDTA (#AM9260G, Ambion, Carlsbad, CA), which was brought to a pH of 7.4 and filter sterilized. Immediately before use, 0.25 mM DTT (CAS #3483-12-3, Fisher Scientific, Waltham, MA), 30 U/mL RNase Out (#10777-019, Invitrogen, Carlsbad, CA), and protease inhibitor cocktail containing AEBSF, Aprotinin, Bestatin, E64, Leupeptin, and Pepstatin A (#1862209, Halt, Thermo Scientific, Rockford, IL), were added to buffer. Centrifugation of whole homogenate (WH) at 500 x g for 10 minutes at 4˚C removed nuclei and cellular debris, yielding pellet, P1 and supernatant, S1. The S1 fraction was passed through a series of nylon filters with successively decreasing pore sizes of 70, 35, and 10 µm (#03-70, #03-35, #03-10, SEFAR, Buffalo, NY). The filtrate was then diluted with 3 volumes of homogenization buffer, and centrifuged at 2000 x g for 15 minutes at 4˚C to yield the synaptoneurosome enriched pellet, P2, and supernatant, S2. Fractions were frozen on dry ice and then stored at -80˚C until further processing. Total RNA was isolated the using guanidine/phenol/chloroform method (#Cs-502, Stat-60, Tel-Test Inc., Friendswood, TX) and a Tekmar homogenizer as per the STAT-60 protocol. RNA concentration was determined by measuring absorbance at 260 nm and RNA quality was assessed by electrophoresis on an Experion Analyzer (Bio-Rad, Hercules, CA) and 260/280 absorbance ratios. All RNA samples had RNA quality indices (RQI) ≥ 7.6 and 260/280 ratios were between 1.97 and 2.06. The preparation of cDNA libraries was conducted following standard protocols using TruSeq RNA Sample Preparation Kit (#RS-122-2001, Illumina, San Diego, CA). Briefly, mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads. The mRNA was then fragmented in the presence of divalent cations at 94˚C. The fragmented RNA was converted into double stranded cDNA and ends polished using T4 and Klenow DNA polymerases, with an adenine base added to the 3' ends followed by ligation of Illumina specific adaptors. The adaptor ligated DNA was amplified with 15 cycles of PCR and purified using QIAquick PCR Purification Kit (#28104, Qiagen, Venlo, Netherlands). Library insert size was determined using an Agilent Bioanalyzer. Library quantification was performed by qRT-PCR assay using KAPA Library Quant Kit (#KK4835, KAPA, Wilmington, MA). RNA-Seq libraries were analyzed using Illumina TruSeq Cluster V3 flow cells and TruSeq SBS Kit V3 (#FC-401-3001, Illumina, San Diego, CA), with six libraries of different indices pooled together in equal amounts loaded on to a single lane at a concentration of 13 pM and sequenced (2 x 100 paired end reads) on a Illumina HiSeq 2000. Sample EE6_P2 was removed from subsequent analyses due to exceedingly high duplication levels.
GEO Accession
GSM1880814