GSM8779190: GFP+ cones, Pde6ccpfl1; Mus musculus; RNA-Seq
Fresh retinas were dissected out of the mouse eyes and dissociated with a papain digestion. A papain/DNase solution (1:20, Worthington Biochemicals, LK003170/LK003176) was made to 100 µL/sample and activated at 37 ºC on a heat block for 30 mins before adding in the dissected retina. Samples were then incubated for 45 mins at 37 ºC and gently triturated with a pipette after to completely dissociate the tissue. 200 µL of inhibitor solution (Earle's balanced salt solution [EBSS; Gibco, 24010-043], 9.5% v/v ovomucoid protease inhibitor [OMI; Worthington Biochemicals, LK003182], 5% v/v DNaseI [Worthington Biochemicals, LK003170]) was added to each sample and was incubated at 37 ºC for 10 mins. The sample was then spun at 1400 × rpm for 5 mins before being resuspended again in 300 µL resuspension solution (EBSS, 10% v/v DNaseI). eBioscience™ Fixable Viability Dye eFluor™ 660 (Invitrogen, 65-0864-14) was used as a dead cell stain and was added at a 1:1000 dilution after resuspension and samples and incubated on ice in the dark for 30 mins. FACS buffer (2% heat-inactivated foetal calf serum [HI-FCS; Fisher Biotec, WS-FBS-AU0-015], 1mM Ethylenediaminetetraacetic acid [EDTA], 1X PBS) was added at 1 mL/sample to wash off the cell stain after incubation and the sample was spun down at 1400 × rpm for 5 mins to collect the pellet. The pellet was resuspended in 500 µL FACS buffer and kept on ice before FACS were performed on the BD FACSMelody™ Cell Sorter to sort GFP+ cones into a 96-well plate for each mouse line. Each well contained 3 µL of lysis buffer and ribonuclease inhibitor. Sorted plates were stored at -80 °C until further processing. After FACS samples were collected, the 96-well plates were sent to the Institute for Immunology and Infectious Diseases (Murdoch University, WA) for further processing. Sample preparation and sequencing was performed using an adapted approach of SMARTseq2 and MARS-seq following methods by Wanjalla et al [Single-cell analysis shows that adipose tissue of persons with both HIV and diabetes is enriched for clonal, cytotoxic, and CMV-specific CD4+ T cells. Cell Reports Medicine. 2021;2(2)]. Cell size, shape or number was not restricted by these approaches. Uniquely tagged primers for reverse transcription and template switching with a pre-amplification step were used in the assay to increase yield and transcript length of the single cell cDNA library. At the reverse transcription stage, cDNAs were tagged with barcodes coupled with a unique molecular identified (UMI) which allowed for multiplexing and increased samples throughput. UMIs also enabled quantification of individual gene expression levels within single cells, thereby reducing technical variability and bias introduced during amplification. Using the KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland) as per the manufacturer's instructions, samples were pooled and amplified. Purified amplicons from the 5' and 3' were combined to equimolar quantities. Indexed libraries were created for sequencing using Truseq adapters and quantification was done using the Kapa universal qPCR library quantification kit (Kapa Biosystems Inc., MA, USA) as per user instructions.