GSM9081592: vehicle control BAs, rep1; Mus musculus; OTHER
Mouse brown adipocytes from a 10 cm dish were washed twice with ice-cold PBS and incubated on ice for 5 minutes in 10 ml swelling buffer (10 mM Tris HCl pH8.0, 2 mM MgCl2, 3mM CaCl2). Cells were then scraped, suspended, filtered through a 100 μm strainer. The cell pellets were resuspended in 250 μl of buffer W (10 mM Tris HCl pH8.0, 5 mM MgCl2, 10 mM KCl, 250 mM sucrose, 1 mM EGTA, 10% (v/v) Glycerol with freshly added 0.5 mM DTT and 0.2 μl/ml RNase inhibitor (SUPERase-In, Invitrogen)). Lysis was performed by adding 10 mL of lysis buffer (buffer W supplemented with 0.5% NP-40 and 0.05% Tween-20), followed by incubation on ice for 5 minutes. The cell suspension was filtered again through a 40 μm strainer, centrifuged, and washed with 10 mL of buffer W. The pelleted cells were then resuspended in freezing buffer (50 mM Tris-HCl, pH 8.0, 40% (v/v) glycerol, 5 mM MgCl₂, 1.1 mM EDTA) supplemented freshly with 0.5 mM DTT and 1 μL/mL RNase inhibitor. Cell permeability was assessed using trypan blue staining. A total of 1–2 million permeabilized cells were snap-frozen in 200 μL of freezing buffer using liquid nitrogen and stored at -80°C. All subsequent steps were performed by the Nascent Transcriptomics Core at Harvard Medical School (Boston, MA). Briefly, run-on reactions were conducted at 37°C. The adenylated 3' adapter was prepared using the 5' DNA Adenylation Kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB), following the manufacturer's instructions with a final concentration of 15% PEG-8000. The reaction was incubated at 16°C overnight. To block unligated adapters, 180 μL of betaine blocking buffer containing 0.6 μM blocking oligo was added to the ligation mixture, followed by incubation at 65°C for 5 minutes and on ice for 2 minutes before streptavidin bead capture. After T4 polynucleotide kinase treatment, the beads were sequentially washed with high-salt, low-salt, and blocking oligo wash buffers before resuspension in the 5' adapter mix. Reverse transcription was performed using SuperScript IV Reverse Transcriptase (ThermoFisher) with 25 pmol of the Illumina TruSeq RP1 primer. The RT product was eluted from the beads and used for library preparation. Libraries were prepared by Nascent Transcriptomics Core at Harvard.