title: GSM8195953: A2a_coc_WD1_4; Rattus norvegicus; RNA-Seq
library_construction_protocol: TRAP was carried out on all 48 brains from the rats that completed the cocaine or saline self-administration procedure, as previously described (Heiman et al., 2014) with modifications (Sanz et al., 2009). Briefly, homogenization buffer (HB) was used as described (Sanz et al., 2009), with heparin omitted. Lysed tissue in HB were centrifuged at 10,000 g for 10 min at 4°C to remove insoluble debris, and the supernatant was used for all subsequent steps. Input samples, equivalent to 12.5% of the initial HB volume, were saved from the supernatant prior to the immunoprecipitation, and RNA was extracted at the same time as TRAP samples. 6.25 μg of two GFP antibodies (bioreactor supernatant, clones 19C8 and 19F7 from the Memorial Sloan-Kettering Institute Monoclonal Antibody Facility) was added to each sample for 4 h at 4C under gentle end-over-end rotation. Samples were transferred to tubes containing 25 μl of Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific; 88803; lot VF298063) and incubated overnight at 4◦C with end-over-end rotation. The following day, beads were washed in high-salt buffer as described (Sanz et al., 2009), and RNA was isolated from the beads and input samples using TRIzol (Thermo Fisher Scientific, 15596018) and the Direct-zol RNA Microprep Kit (Zymo Research, R2062). RNA concentration was determined using the Quant-iT RiboGreen RNA Assay (Thermo Fisher Scientific, R11490) using a CLARIOstar Plus plate reader (BMG Labtech). TRAP IP controls with either no antibody or using wild-type tissue resulted in no measurable RNA based on the RiboGreen assay and only trace amounts of RNA detected using qRT-PCR. RNA-seq libraries were profiled on a 4200 Tapestation (Agilent)