GSM7110364: astrocyte, control, TRAP, 20_2; Mus musculus; RNA-Seq
TRAP was carried out on 6 control and 6 ethanol treated 100 mm dishes as previously described (Heiman et al., 2014) with modifications (Sanz et al., 2009). Briefly, homogenization buffer (HB) was used as described in Sanz et al, with heparin omitted. Lysed cells in HB were centrifuged at 10,000 g for 10 min. at 4° C to remove insoluble debris and the supernatant used for all subsequent steps. Input samples were saved from the supernatant equivalent to 5% of the initial HB volume. 50 mg of two GFP antibodies (bioreactor supernatant, clones 19C8 & 19F7 from the Memorial Sloan-Kettering Institute Monoclonal Antibody Facility) were added to each sample for 4h at 4° C under gentle end-over-end rotation. Samples were transferred to tubes containing 200 mL 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 in Sanz et al, 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 libraries were generated using the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit according to the manufacturer's protocol