GSM8265165: RC77N/E 10nM 1a25(OH)2D3 treated Rep 1; Homo sapiens; RNA-Seq
Before handling RNA, surfaces and instruments underwent treatment with RNase Zap (Ambion AM9780, Austin, U.S.A.). RNA extraction from cultured cells followed the Rneasy Mini protocol (Qiagen 7414, Hilden, Germany). Initially, cells were rinsed with PBS, then lysed with buffer RLT and 10 μL/mL β-mercaptoethanol, utilizing 350 μL per T25 flask or 600 μL per 10 cm plate. Subsequently, the Rneasy Mini protocol, inclusive of on-column incubation with Rnase-free Dnase (Qiagen, Cat: 79254), was adhered to. Post washes, RNA was eluted using 30 μL Rnase-free water, and its concentration was determined employing a Nanodrop spectrophotometer. RNA samples were stored at −80 °C, with each sample being isolated and utilized individually in triplicate. RNA sequencing was performed by the City of Hope Integrative Genomics Core facility using RNA sequencing libraries prepared with Kapa RNA mRNA HyperPrep kit (Kapa Biosystems, Wilmington, U.S.A., Cat KR1352) according to the manufacturer's protocol. The final libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit and quantified with Qubit. Sequencing was performed on Illumina HiSeq 2500 with the single read mode of 51 cycles. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis.