title: GSM4182005: Petro12 Input; Petrotilapia sp. 'chitimba'; RNA-Seq
library_construction_protocol: Whole brains were removed, flash frozen, and stored at -80C. Frozen brains were homogenized in 2mL homogenization buffer on ice [10 mM HEPES, 150mM KCl, 5mM MgCl2 in RNAse-free water, plus 5uL of 1M DTT, 1/2 EDTA-free protease inhibitor tablet (Roche 11836170001), 5uL RNAsin (Promega N2611), 5uL cycloheximide (100mg/mL stock in DMSO), 10uL phosphatase inhibitor cocktail (ThermoScientific 78440), and 2uL of Calyculin A (1mg/mL in ethanol stock] per 2mL added 1 hour prior]. The lysate was then centrifuged at 2000xg for 10 min at 4C. The supernatant from each tube was transferred to new tubes and 140uL of 10% NP40 and 140uL DHPC (300 mM stock in chloroform). The tubes were mixed briefly and then incubated for 2 min on ice. The tubes were then spun at Rmax for 10 min at 4C and the supernatant was collected. For the input RNA sample, 50uL of this supernatant was added to 350uL of buffer RLT (Qiagen RNeasy Micro Kit) and stored on ice until purification. For the IP samples, immunoprecipitation was performed using Protein A Dynabeads (Life Technologies 10001D) loaded with pS6 antibody (Cell Signal 2215S). Briefly, we rinsed the pS6-loaded magnetic beads 2x in 0.15M KCl buffer and distributed the beads into two microcentrifuge tubes for each IP. After the second wash, the 0.15M KCl buffer was removed and then ~1mL of clarified supernatant was added to each tube and mixed to resuspend the beads. The tubes were incubated with end-over-end mixing for 10 min at 4C, which allows for antibody binding. After a brief spin, the tubes were placed on the magnet, and the supernatant was removed. The beads were then rinsed 4x in 0.9mL of cold 0.35M KCl buffer (10mM HEPES, 350mM KCl, 5mM MgCl2, 1% NP40, plus the following supplements added per 4mL: 5uL 1M DTT, 10uL RNAsin, 5uL cycloheximide, and 2uL Calyculin A). After the 3rd wash, the samples incubated at room temperature for 5 min, which allows for the dissociation of some of the non-specific RNA. After the 4th wash, the supernatant was removed, and the beads were resuspended in 350uL of buffer RLT and incubated on ice for 5 min. RNA from both the input and IP samples was purified using the Qiagen RNeasy Micro Kit (Qiagen 74004). RNA from both the input and IP samples was purified using the Qiagen RNeasy Micro Kit (Qiagen 74004). cDNA synthesis was performed using the SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio 634888). We used 2uL of input RNA and the 10.5uL of the remaining IP RNA for cDNA synthesis and the maximum number of PCR cycles (18) during the cDNA amplification step. Library preparation was performed using NEBNext Ultra II FS DNA library Prep Kit for Illumina (NEB E7805S). Library preparation was performed at the Molecular Evolution core facility at GT using NEBNext Ultra II FS DNA library Prep Kit for Illumina (NEB E7805S). Either 1ng (IP samples), or 5ng (Input samples) of cDNA were used as a starting material for the library preparation. The samples were pooled by equimolar normalization and sequenced on together in a single run on an Illumina NextSeq 500 instrument with 76 cycles (single-end).
identifiers.namespace: GEO
identifiers.id: GSM4182005