title: GSM4411755: habenula, sample 3; Mus musculus; RNA-Seq
library_construction_protocol: Mice were transcardially perfused with an ice-cold choline cutting solution (110 mM choline chloride, 25 mM sodium bicarbonate, 12 mM D-glucose, 11.6 mM sodium L-ascorbate, 10 mM HEPES, 7.5 mM magnesium chloride, 3.1 mM sodium pyruvate, 2.5 mM potassium chloride, 1.25 mM sodium phosphate monobasic, saturated with bubbling 95% oxygen/5% carbon dioxide, pH adjusted to 7.4 using sodium hydroxide). Brains were rapidly dissected out and sliced into 200 µm thick coronal sections on a vibratome (Leica Biosystems, VT1000) with a chilled cutting chamber filled with choline cutting solution. Coronal slices containing the habenula 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 (Thermo Fisher Scientific Cat. # 14170112, supplemented to final content concentrations: 138 mM sodium chloride, 11 mM D-glucose, 10 mM HEPES, 5.33 mM potassium chloride, 4.17 mM sodium bicarbonate, 2.12 mM magnesium chloride, 0.441 mM potassium phosphate monobasic, 0.338 mM sodium phosphate monobasic, saturated with bubbling 95% oxygen/5% carbon dioxide, pH adjusted to 7.35 using sodium hydroxide) and kept on ice until dissections were completed.  Dissected tissue chunks for each sample were pooled for each hemisphere for the subsequent dissociation steps. Tissue chunks were first mixed with a digestion cocktail (dissociation media, supplemented to working concentrations: 20 U/ml papain, 0.05 mg/mL DNAse I) and incubated at 34 °C for 90 min with gentle rocking. The digestion was quenched by adding dissociation media supplemented with 0.2% BSA and 10 mg/ml ovomucoid inhibitor (Worthington Cat. # LK003128), and samples were kept chilled for the rest of the dissociation procedure. Digested tissue was collected by brief centrifugation (5 min, 300 g), re-suspended in dissociation media supplemented with 0.2% BSA, 1 mg/ml ovomucoid inhibitor, and 0.05 mg/mL DNAse I. Tissue chunks were then mechanically triturated using fine-tip plastic micropipette tips of progressively decreasing size. The triturated cell suspension was filtered through a 40 µm cell strainer (Corning 352340) 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 (Sigma D1556). 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). Suspensions were kept chilled until the cells were flowed into the microfluidic device. Libraries were prepared and indexed following the protocols referenced above, and sequencing-ready libraries were stored at -80 °C. Libraries from different samples were pooled and sequenced on an Illumina NextSeq 500 (High Output v2 kits). Libraries were constructed following the protocols described in Zillionis et al. (2017) for inDrop v3 hydrogels. 2 libraries were pooled for sequencing 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.
attributes.tag: GEO Accession
attributes.value: GSM4411755