title: GSM3938221: Dorsal Raphe Nucleus, Control, Batch 2, Set 5; Mus musculus; RNA-Seq
library_construction_protocol: Mice were transcardially perfused with an ice-cold choline cutting solution containing neuronal activity blockers (10 µM (R)-CPP, 1 µM tetrodotoxin). Brains were rapidly dissected out and sliced into 250 µm thick coronal sections on a vibratome in a chilled cutting chamber filled with choline cutting solution. Coronal slices containing the DRN were then transferred to a chilled dissection dish containing a choline-based cutting solution for microdissection. Dissected tissue chunks were transferred to cold HBSS-based dissociation media with an inhibitor cocktail (0.9 mM kynurenic acid, 10 µM (R)-CPP, 1 µM tetrodotoxin, 10 µM triptolide, 5 µg/ml actinomycin D, 30 µg/ml anisomycin) and kept on ice. Dissected tissue chunks for each sample were pooled into a single tube for dissociation. Tissue chunks were mixed with a digestion cocktail (dissociation media with 20 U/ml papain, 1 mg/ml pronase, 0.05 mg/mL DNAse I, 10 µM triptolide, 5 µg/ml actinomycin D, 30 µg/ml anisomycin) and incubated at 34 °C for 90 min with gentle rocking. Digestion was quenched by adding dissociation media with 0.2% BSA and 10 mg/ml ovomucoid inhibitor, and samples were kept chilled for the rest of the dissociation procedure.  Tissue chunks were then mechanically triturated using fine-tip plastic micropipette tips of progressively decreasing size. Triturated cell suspension was filtered in two stages using a 70 µm cell strainer and 40 µm pipette tip filter and washed in two repeated centrifugation (5 min, 300 g) and re-suspension steps to remove debris before a final re-suspension in dissociation media containing 0.04% BSA and 15% OptiPrep. Cell density was calculated based on hemocytometer counts and adjusted to approximately 100,000 cells/ml. Single-cell encapsulation and RNA capture on the InDrop platform was performed at the Harvard Medical School ICCB Single Cell Core using v3 chemistry hydrogels based on previously described protocols (Zilionis et al., 2017). Libraries were constructed following the protocols described in Zillionis et al. (2017) for inDrop v3 hydrogels. 4 libraries were pooled for sequencing (2 sequencing runs per set) and demultiplexed using library barcodes (see README file). R1 is the biological read, R2 is the first half of the hydrogel barcode, R3 is the library index, and R4 contains the second half of the hydrogel barcode, UMI, and fraction of the polyA tail.