title: GSM7507874: Control left eyes - Rep 3; Gallus gallus; RNA-Seq
library_construction_protocol: Choroidal cells were isolated as described previously (Coulombe & Nishi, 1991) with minor modifications: Choroids were cut into small pieces, and digested with collagenase [Worthington; 2mg/ml in HBSS containing Ca++ and Mg++; 2ml/ sample] for 20 min at 37°C. Following centrifugation at 834 x g at 4°C for 15 min, the supernatants were removed and samples were digested with 2 ml trypsin (Sigma; 0.2% in PBS for 20 min at 37°C). Choroids were again centrifuged as described above, trypsin removed, and replaced with another 2ml of collagenase solution (2 mg/ml in HBSS) and incubated at 37°C with occasional trituration using a glass Pasteur pipette until the digest was able to pass freely through the glass Pasteur pipette. Following a second centrifugation as described above, supernatants were removed and the pelleted choroidal digests were resuspended in PBS + 0.04% BSA (1 ml/sample). The dissociated cell suspensions were passed through small, individual 105 μm metal mesh screens held in place with 25 mm Swinnex filter holders (MilliporeSigma, Burlington, MA ) using  5 cc syringes (6 filters and 6 syringes in total).  Each filtrate was collected in 4 ml snap cap tubes. Each filter was rinsed with an additional 1 ml PBS + 0.04% BSA and added to each original filtrate.  Each cell sample was then passed through a 40 μm pipette tip cell strainer (FLOWMI; SP Bel-Art, Wayne, NJ).  Living and dead cells were identified by simultaneously staining with green-fluorescent calcein-AM and red-fluorescent ethidium homodimer-1, respectively. 10xChromium single cell libraries were prepared according to the standard protocol outlined in the manual. Briefly, sorted single cell suspension, 10x barcoded gel beads, and oil were loaded into Chromium™ Single Cell A Chip to capture single cells in nanoliter-scale oil droplets by Chromium™ Controller and to generate Gel Bead-In-EMulsions (GEMs). Full length cDNA libraries were prepared by incubation of GEMs in a thermocycler machine. GEMs containing cDNAs were broken and all single cell cDNA libraries were pooled together, cleaned using DynaBeads MyOne™ Silane beads (Fisher PN 37002D), and pre-amplified by PCR to generate sufficient mass for sequencing library construction. Sequencing libraries were constructed by following the steps: cDNA fragmentation, end repair & A-tailing, size selection by SPRIselect beads (Beckman Coulter, PN B23318), adaptor ligation, sample index PCR amplification, and a repeat of SPRIselect beads size selection. The final constructed single cell libraries were sequenced with a total reads per cell targeted for a minimum of 50,000.