Anti mouse igg1 microbeads

Single cell libraries were generated by minor modifications of methods developed for macaque retina^{12 (link)}. Briefly, a ~1.5 mm diameter circular region centered on the foveal pit was dissected from the retina, and peripheral retinal pieces were pooled from all retinal quadrants. Dissected tissues were digested with papain (Worthington, LS003126) for 30 min at 37 °C. Following digestion, samples were dissociated and triturated into single cell suspensions with 0.04% bovine serum albumin (BSA) in Ames. Dissociated cells from digested peripheral retinas were incubated with CD90 microbeads (Miltenyi Biotec, 130-096-253; 1 ml per 10⁷ cells) to enrich RGCs or with anti-CD73 (BD Biosciences, clone AD2; 5 ml per 10⁷ cells) followed by anti-mouse IgG1 microbeads (Miltenyi Biotec, 130-047-102; 10 ml per 10⁷ cells) to deplete rods. Incubations were at room temperature for 10 min. CD90 positive cells or CD73 negative cells were selected via large cell columns through a MiniMACS Separator (Miltenyi Biotec). Foveal samples were used without further processing. Single cell suspensions were diluted to 500–1800 cells/µL in 0.04% BSA/Ames for loading into 10X Chromium Single Cell v2 or v3 Chips. Following collection, cDNA libraries were prepared following the manufacturer’s protocol, and sequenced on the Illumina HiSeq. 2500 (Paired end reads: Read 1, 26 bp, Read 2, 98 bp).

Bovine blood samples were collected by veterinarians and PBMCs were separated from the blood samples using density-gradient centrifugation on Percoll (GE Healthcare, Little Chalfont, UK). The experiments using bovine blood samples were approved by the Ethics Committee of the Faculty of Veterinary Medicine, Hokkaido University (approval numbers: 17-0014 and 17-0024). These experiments were carried out in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357). To isolate CD14⁺ cells from PBMCs, PBMCs were cultured with anti-bovine CD14 monoclonal antibody (mAb) (CAM36A; WSU Monoclonal Antibody Center, Pull-man, WA, USA) for 30 min at 4 °C. Cells were then incubated with anti-mouse IgG₁ MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4 °C. After incubation, CD14⁺ cell sorting was conducted using an autoMACS Pro (Miltenyi Biotec) according to the manufacturer’s protocol. The purity of the CD14⁺ cells (> 90%) was confirmed using FACS Verse (BD Biosciences, San Jose, CA, USA). Cells were cultured in RPMI 1640 medium (Sigma-Aldrich) containing 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin (Thermo Fisher Scientific), 100 μg/mL streptomycin (Thermo Fisher Scientific), and 2 mM l-glutamine (Thermo Fisher Scientific) using 96-well plates (Corning Inc., Corning, NY, USA).