E-MTAB-6058:Sample 47_p
NextSeq 500 paired end sequencing; Single cell RNA-seq of isolated human foetal L/M-opsin cone photoreceptor populations labelled using the AAV2/9.pR2.1.GFP reporter
Single cell RNA-seq of isolated human foetal L/M-opsin cone photoreceptor populations labelled using the AAV2/9.pR2.1.GFP reporter
Sample 47_p
pR2.1.GFP+ were isolated via FACS and separated using a medium-sized (10-17µm cell diameter) integrated fluidic circuit (IFC; Fluidigm), using the Fluidigm C1 system. Prior to loading on the IFC, cell viability was assessed using a standard trypan blue exclusion test before resuspending cells at a concentration of 667cells/µl. 3µl total sample volume was loaded onto IFC and cell were imaged via phase-contrast microscopy to check for the single cell status of each chamber.  Human foetal eyes were obtained from the Joint Medical Research Council UK (grant # G0700089)/Wellcome Trust (grant # GR082557) Human Developmental Biology Resource (http://www.hdbr.org/)  Intact human foetal retina were cultured free floating in 12 well plates with retinal differentiation media (RDM) containing DMEM-F12 (Gibo), Glutamax (Gibo), 1x N2 and 1x B27 neural supplements (Invitrogen), 10% FBS (Invitrogen) and 1.5x penicillin/streptomycin (Invitrogen). Cell culture media was changed every 2 days. AAV2/9.pR2.1:GFP production and application: 293T cells were cultured in D10 medium containing DMEM+Glutamax, 10% (v/w) FBS and 1x pencillin/streptomycin in 150cm plates and grown to 80% confluency. pd10/pR2.1.L/Mopsin-eGFP construct, pHGT1 helper vector, AAV2/9 caspid were transfected into 293T cells using polyethylenimine (PEI), before harvesting cells after 72 hours and performing four thaw-vortex-freeze cycles for viral release. pR2.1.L/M-opsin promoter was originally generated by the Jeremy Nathans group (Wang et al., 1992). Virus lysate underwent benzonase treatment and was prepared for purification through multiple centrifugation steps and sequential filtering with 5µm, 0.45µm and 0.22µm PES membranes. Virus was purified via ion exchange chromatography using AKTA Prime (GE Life Sciences) and concentrated using a Vivaspin 4 concentrator (Vivaproducts). Viral genomic titer was determined through qRT PCR and the virus titer used in this study ranged from 1.48x1013-1.01x1014. Virus was added to the human foetal retinal explant at a MOI of 40,000-45,000 through addition to culture media. Human foetal explant was cultured for 7 days to allow for pR2.1:GFP expression to be visualised. On day 7, human foetal retinal explant was dissociated enzymatically into a single cell suspension using the papain dissociation system (Worthington Biochemical, Lorne Laboratories) following manufacturer's protocol. The 15pcw foetal retinal sample was cut into smaller pieces (5mm x 5mm) before the addition of papain (approx. 100µl per piece). 70µm cell strainers were used to ensure cell single density of cell suspension. For GFP cell isolation, cells were resuspended in a final volume of 500µl blocking solution (1% (w/v) BSA in PBS). The FACSAria III Cell Sorter and the MoFlo XDP Cell Sorter (BD Biosciences) were used to isolate GFP+ cells. Cell populations (GFP+ and GFP-) were collected into eppendorf tubes containing 500µl of 50% v/v FBS (DMEM). After sorting, cell populations were collected via centrifugation for 20 minutes at 200 x g; the supernatant was subsequently removed and RNA extraction was performed on cell pellets. Single cells are separated into chambers of IFC by Fluidigm C1 system via microfluidics, before cell lysis and RNA amplification is performed. Cell harvest is collected and processed for sequencing. Cell harvests were subject to the SMART-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories) and cDNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina).
UCL GOS Institute of Child Health
E-MTAB-6058:Sample 47_p
Experimental Factor: single cell identifier
Cell 47