Newborn FVB/N strain mouse lenses were dissected into capsules containing adhering epithelial cells and fiber cells. Epithelial and fiber cell fractions were each pooled into three biological replicates for a total of six samples, each containing tissue from 8 lenses. Total RNA was extracted using mirVana™ miRNA isolation kit (# AM1560, ThermoFisher Scientific). Small RNA was isolated from total RNA by size-selection and NEBNext Multiplex Small RNA Library Prep Kit was used for 50bp-single-ended sequencing to yield ~5 million reads per sample.
Four whole lenses were harvested from miR-26 triple knockout (TKO) mice at two stages (Day 5 and 20 weeks). For 20-week-old mice we selected lens exhibiting cataract (C) and TKO mice without cataract upon visual inspection. RNA extraction was performed using RNEasy Mini Kit (Qiagen cat. 74104) following the manufacturer’s instructions. The RNA Integrity Number (RIN) was determined using an Agilent 2100 Bioanalyzer and samples with RIN > 7 were used for sequencing. RNA passing in-house quality control were sent to Novogene (Sacramento, CA, USA) for mRNA library preparation and sequencing using NovaSeq PE150 with approximately 30 million reads per sample.
Four whole lenses were harvested from miR-26 triple knockout (TKO) mice at two stages (Day 5 and 20 weeks). For 20-week-old mice we selected lens exhibiting cataract (C) and TKO mice without cataract upon visual inspection. RNA extraction was performed using RNEasy Mini Kit (Qiagen cat. 74104) following the manufacturer’s instructions. The RNA Integrity Number (RIN) was determined using an Agilent 2100 Bioanalyzer and samples with RIN > 7 were used for sequencing. RNA passing in-house quality control were sent to Novogene (Sacramento, CA, USA) for mRNA library preparation and sequencing using NovaSeq PE150 with approximately 30 million reads per sample.