title: Illumina HiSeq 2500 paired-end Sequencing of KAS23061858 (KAE23678080)
design: Reference sequence : mouse mm10
library_construction_protocol: Primary renal proximal tubule cells were isolated from C57BL/6J mice sacrificed at 8 weeks according to the Institutional Animal Care and Use Committee IACUC of GIST ethics committee guidelines. After removing medulla, renal cortex cells were dissociated into single cells using a tissue dissociation kit Multi Tissue Dissociation Kits, Miltenyi Biotec. Cells were incubated on the collagen Collagen I bovine, ThermoFisher coated plated in 5% CO2 at 37degree Celsius and grown in Renal Epithelial Cell Medium, which was composed of Fetal Calf serum 0.05ml/ml, Epidermal Growth Factor 10ng/ml, Insulin 5microgram/ml, Epinephrine 0.5 microgram/ml, Hydrocortisone 36 ng/ml, Triiodo-L-thyronine 4pg/ml, Transferrin 5microgram/ml Renal Epithelial Cell Growth Medium 2, PromoCell. Cells were subcultured up to 2 passages which are optimized passages for transfection. Primary renal proximal tubule cells were transfected with 0610005C13Rik-siRNA #,ThermoFisher or Silencer??Select Negative Control No. 1 siRNA #,ThermoFisher using Lipofectamine??RNAiMAX #,ThermoFisher. Transfected cells were harvested 24 hours after transfection and total RNA was isolated using RNA isolation kit RNeasy Mini kit, QIAGEN. After library preparation, RNA sequencing Illumina TruSeq; 100-bp paired-end reads was performed at Macrogen, Seoul, South Korea.