title: 10x single-cell RNA-seq of HVC from Bengalese finch
design: Three adult male Bengalese finches were euthanized by isoflurane overdose and decapitated. Brains were transferred to ice cold artificial cerebrospinal fluid (124.7 mM choline chloride, 3 mM MgSO4, 0.5 mM CaCl, 2.5 mM KCl, 25 mM NaHCO3, 35 mM glucose) bubbled with Carbogen then sliced from the posterior in a coronal orientation at 260 m on a vibratome. Sections containing HVC or RA were transferred to bubbled ACSF and allowed to recover for 15 minutes. HVC, NC, RA, and ARCO when then then manually microdissected. HVC and RA were identified by their known anatomical position and their dense myelination. NC was collected just ventral but not directly adjacent to HVC  within the nidopallium. ARCO was collected just lateral to but not directly adjacent to RA wtihin the arcopallium. For each bird, two to three slices of each region from each hemisphere were collected and transferred to tubes containing 1 mL bubbled ACSF. 50 L 20 mg/mL pronase (Sigma) was added to each tube, tubes were transferred to 37 C in a shaking incubator for 45 minutes. After pronase incubation, samples were passed through three rounds of glass Pasteur pipettes fire-polished to progressively smaller pore sizes (600, 300, and 150 m). After dissociation samples were spun down at 100xg for 1 minute at 4 C and washed twice in ACSF containing 1% fetal bovine serum (FBS), resuspended in ACSF/1% FBS and 1 ng/mL DAPI, filtered through 40 m FlowMi filters (Scienceware). Samples were then flow sorted to exclude cells with high DAPI intensity. After collection cells were spun down at 100xg for 1 minute at 4 C and resuspended in 50 L PBS with 0.04% bovine serum albumin, counted, then adjusted to a concentration of 230-400 cells / L. Samples were then transferred to the Gladstone Genomics Core where they were loaded into Chromium (10X Genomics) with the Chromium Single Cell 3 Library & Gel Bead Kit v2 (10X Genomics). Libraries were constructed according to manufacturers instructions with separate i7 indices then pooled. An initial round of sequencing on an Illumina Miseq was used to determine approximate cell numbers in each samples, then the samples were sequenced on four lanes of an Illumina Hiseq 4000 for 25 cycles R1 and 96 cycles R2.