Total RNA was extracted from the samples using the Qiagen RNeasy mini kit reagent (74104; Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The quantity and quality of extracted RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized with an appropriate amount of RNA using the ReverTra AceTM qPCR RT Kit (TOFSQ-101; TOYOBO Co., Ltd., Osaka, Japan), according to the manufacturer’s recommendations. After RNA denaturation at 65 °C for 5 min, 1 μg of total RNA was diluted in 10 μL of reaction mixture containing 2 μL 5X RT buffer, 0.5 μL enzyme mix, 0.5 μL Primer mix, and water. The reaction mixture was incubated at 37 °C for 15 min. The cDNA product was further diluted four-fold with RNase-free water and used directly for real-time PCR.
The amplified cDNA samples were obtained in the library preparation step using Chromium Single Cell 5′ Library & Gel Bead Kit v1.1 (scRNA-Seq) [19 (link)] and Chromium Single Cell 3′ Library & Gel Bead Kit v3 (snRNA-Seq), according to the manufacturer’s recommendations.
The amplified cDNA samples were obtained in the library preparation step using Chromium Single Cell 5′ Library & Gel Bead Kit v1.1 (scRNA-Seq) [19 (link)] and Chromium Single Cell 3′ Library & Gel Bead Kit v3 (snRNA-Seq), according to the manufacturer’s recommendations.