title: Human pluripotent stem cell-derived, ROR1-expressing lens epithelial cells
organism: Homo sapiens
description: Protocols: ROR1-expressing lens epithelial cells (ROR1e LECs) were harvested from CA1 human embryonic stem cells undergoing directed differentiation towards lens cells. ROR1e LECs were collected via TrypLE on day 16 of differentiation, and cultured for a further 7 days in LEC medium (Murphy et al, Development. 2018) before being harvested using TrypLE and processed for single cell RNA-sequencing. CA1 human embryonic stem cells were obtained from A. Nagy (Adewumi et al. 2007). Approval for their use was obtained from the Western Sydney University Human Research Ethics Committee (Australia). CA1 cells were maintained on Matrigel-coated plates (Corning, Sydney, Australia) in mTeSR1 (StemCell Technologies, Sydney, Australia) as previously described (O'Connor, Kardel et al. 2008). Generation of heterogeneous differentiation cultures containing lens cells occurred in: DMEM:F12 (Thermo Fisher Scientific, Sydney Australia) containing 500 ng/mL noggin and 10 nM SB431542 (6 days); followed by DMEM:F12 containing 20 ng/mL BMP4, 20 ng/mL BMP7 and 100 ng/mL FGF2 [2]. Growth factors were sourced from Miltenyi Biotec and Peprotech (Sydney, Australia). ROR1e LECs were harvested using TrypLE (Thermo Fisher Scientific), filtered through a 40 m cell strainer (Interpath Services, Sydney, Australia), centrifuged (300g, 5 minutes) and counted, then resuspended in medium containing 10 ng/mL FGF2, 5 ng/mL EGF and TGFα, 10 ng/mL HGF, 10 ng/mL IGF1, 10 g/mL insulin and 10 ng/mL PDGF-AA (Murphy et al. Development. 2018). After 7 days, ROR1e LECs were harvested using TrypLE and immediately processed for scRNA-seq. ROR1e LECs were processed immediately after collection using a 10X Genomics single cell 3' mRNA-prep kit as per the manufacturer instructions. Library construction was performed using a 10X Genomics single cell 3' mRNA-prep kit as per the manufacturer instructions.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-11-16T09:22:55Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-06-04T12:11:18Z
attributes.tag: External Id
attributes.value: SAMEA6873237
attributes.tag: INSDC center name
attributes.value: Western Sydney University
attributes.tag: INSDC first public
attributes.value: 2020-11-16T09:22:55Z
attributes.tag: INSDC last update
attributes.value: 2020-06-04T12:11:18Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9178:Human pluripotent stem cell-derived, ROR1-expressing lens epithelial cells
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: CA1
attributes.tag: cell type
attributes.value: lens epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: phenotype
attributes.value: ROR1 positive
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-9178:Human pluripotent stem cell-derived, ROR1-expressing lens epithelial cells
attributes.tag: scientific_name
attributes.value: Homo sapiens