Amplicon-Seq of mus musculus: adult 90 days retina
Purified amplicons were quantified using Qubit 1x dsDNA high sensitivity kit (Q33231), and 750-1000ng total DNA was used for NGS library preparation. NEBNext Ultra II DNA Library Prep kit (E7103) was used to ligate Illumina adapters onto DNA followed by size selection for final library size of ~280bp. Libraries were multiplexed by adding 8bp indexes (E6440) during the amplification step. Library quality was assessed by Tape Station 4200 HS D100 (5067-5584) and quantified using Qubit HS D1000 before normalizing all libraries to 4nM concentration with a final loading concentration of 10pM after pooling. Sequencing was performed on an Illumina MiSeq instrument with v3 flow cell and a 150-cycle reagent kit (MS-102-3001) with 1x130 single reads to achieve the desired coverage.