GSM8487260: lens epithelium,wildtype,biorep3; Mus musculus; RNA-Seq
The lens epithelial cells were isolated based on a previously published procedure. 36 Mice at 1- month-old were used for lens primary epithelial cell culture. Briefly, lenses were dissected from enucleated eyeballs of CO2-euthanized mice in Advanced DMEM/F-12 media (Gibco Invitrogen, Cat# 12634-010, Waltham, MA). The lenses were cleaned by removing the surrounding tissues and were incubated in 0.05% Trypsin-EDTA (Gibco, Cat# 25300-054) at 37°C for 10 minutes. Lens capsules were peeled off and transferred into 2 U/mL dispase (Sigma, Cat# D4693) for 5 minutes at 37°C. Then equal volumes of 10TrypLE (Gibco, Cat# A12177- 02) were added for 10 minutes. The suspended epithelial cells from lens capsules were spun down at 1000 rpm for 5 minutes, and the cell pellet was resuspended in PBS. Collected lens epithelial cells pooled from three WT littermates and four Cx50KO littermates were used for single-cell RNA sequencing in the Functional Genomics Laboratory (FGL) at UC Berkeley. Single-cell cDNA libraries were prepared from the isolated cells using the Chromium Single Cell 3′ System according to the manufacturer's instructions. The protocol from 10x employed v3.1 chemistry with the following modification was used: the cell suspension was directly added to the reverse transcription master mix, along with the appropriate volume of water to achieve the  7 approximate cell capture target. Completed libraries were sequenced on Illumina 100PE Collected lens epithelial cells pooled from three WT littermates and four Cx50KO littermates were used for single-cell RNA sequencing in the Functional Genomics Laboratory (FGL) at UC Berkeley. Single-cell cDNA libraries were prepared from the isolated cells using the Chromium Single Cell 3′ System according to the manufacturer's instructions. The protocol from 10x employed v3.1 chemistry with the following modification was used: the cell suspension was directly added to the reverse transcription master mix, along with the appropriate volume of water to achieve the  7 approximate cell capture target. Completed libraries were sequenced on Illumina 100PE HiSeq4000 to produce 10x recommended reads.