title: GSM9117336: PVH cells, female, sample 3, drop 1, RT-rxn A, lib. 1; Mus musculus; RNA-Seq
library_construction_protocol: C57BL/6J or Sim1-Cre::R26-LSL-EGFP-L10a mice aged 6-12 weeks were sacrificed between 9 am – 12 pm by rapid decapitation immediately after removal from the home cage. Brains were rapidly extracted and chilled in DMEM/F12 media slush. Next, brains were placed ventral side up in a chilled stainless steel brain matrix (Roboz Surgical Instrument Co.: SA-2165) and 1 mm coronal sections of the hypothalamus were collected. The PVH was then microdissected under a fluorescent stereoscope. For each sample preparation, 4-6 male or female mice were pooled. Pooled tissue samples were digested in papain solution with DNAse for 1hr at 37C with gentle agitation, according to a published protocol (Saxena A, et al.) except that an entire vial of papain was used in a 1.6mL final reaction volume. Digestion was stopped by dilution with “EBSS #2” 56, with 10% fetal bovine serum used in place of the ovomucoid protease inhibitor. Tissue was centrifuged at 300×g for 5 min, re-suspended in the modified EBSS #2, then gently triturated using a series of 5 Pasteur pipettes with tips fire-polished to incrementally smaller openings. The resulting cell suspension was divided between two microfuge tubes, diluted with Drop-seq buffer (Macosko et al., 2015) to a volume of 1.5mL per tube. Cells were then washed twice by centrifugation at 300×g for 5 min, followed by re-suspension in Drop-seq buffer. Drop-seq buffer consisted of: 5% weight/volume trehalose; 1x Hank's Buffered Salt Solution (magnesium- and calcium-free); 2.13mM MgCl2; 2mM MgSO4; 1.26mM CaCl2; 1mM glucose; 0.01% bovine serum albumin (molecular biology grade). Cells were filtered through a 20um mesh, diluted to 220 cells/uL based on cell concentration estimates from a hemacytometer, and then kept on ice until use. Drop-seq was performed as in the reference (Macosko et al., 2015) but with the following modifications: first, flow rates of 2.1 mL/hr were used for each aqueous suspension, and 12 mL/hr for the oil. Second, libraries were sequenced on the Illumina NextSeq 500, using 1.7 pM in a volume of 1.2 mL HT1 and 3 mL of 0.3 μM Read1CustSeqB.