title: GSM7047222: Coneal_epithelium_snRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Human limbus tissue was extracted during cataract surgery and flash frozen on LN2 for preservation of gene expression profiles of the cell sub-types within the ocular surface epithelium. Healthy human limbal tissue was collected from consenting cataract patients under the Royal Victorian Eye and Ear Hospital Human Research Ethics Committee approved study (ID:10/954H). Limbus tissue samples were snap frozen within 2 minutes of excision using LN2 were pooled and nuclei isolation was carried out using the Nuclei Isolation Kit: Nuclei EX Prep (Sigma, NUC101) as described previously (PMID 28846088). All steps were carried out on ice. Briefly, tissue samples were homogenized using a glass Dounce grinder in 2ml of ice-cold Nuclei EZ lysis buffer (Sigma N3408, with RNase inhibitor). Samples were transferred to a separate tube on ice while the glass grinder was rinsed with 2mL of Nuclei EZ lysis buffer. This rinse was pooled into the sample for a total of 4mL and then incubated on ice for 5 min. Nuclei were centrifuged at 500 x g for 5 min at 4oC and washed with ice-cold EZ buffer, followed by a second wash in nuclei suspension buffer (NSB) consisting of PBS (Gibco 14190-144), 1% w/v BSA (Sigma, A9576) and 0.2U/uL RNase inhibitor (Takara, 2313A). Isolated cell nuclei from cornea limbal samples were resuspended in 200uL NSB containing 5ug/mL DAPI (ThermoFisher Scientific, D1306). Nuclei integrity was visually inspected under an inverted phase microscope. Isolated nuclei were then filtered through a 35um nylon mesh cell strainer for nuclei sorting using a FACSAria Fusion sorter (BD Biosciences, 70um nozzle) at a flow rate between 1-3 to achieve no more than 1000 events per second. Nuclei, defined as DAPI-positive singlets, were sorted into microtubes containing 35uL NSB. Attempts to count Trypan Blue-positive stained nuclei either prior to soring or immediately after sorting indicated that the number of nuclei was too low to accurately count without sacrificing a large portion of the cornea samples. Therefore, nuclei were resuspended in the minimal volume required for each step in the isolation procedure without counting or dilution. The average percentage recovery from the FACSAria Fusion sorter was determined from other samples to be 70%, so the final nuclei count for sequencing was calculated by multiplying the number of DAPI+ nuclei sorted x 0.7. Sorted nuclei were pelleted by centrifugation (500 x g, 2 min, 4C), then carefully resuspended in either an appropriate volume of NSB to yield 100-200k nuclei per mL or a minimum of 20uL NSB (whichever volume was larger). Library Construction was performed using the Chromium Next GEM Single Cell 3' Library & Gel Bead kit v3.1 (10x Genomics (PN-1000121) with 18 complementary DNA pre-amplification cycles and sequencing on one-high output lane of the NextSeq 500 (Illumina). Sequencing was performed with libraries pooled and loaded in a single lane of an MGISEQ2000-RS sequencer (MGI Tech Co Ltd). snRNA-seq