GSM9082476: WT2, CCD-1, dDAVP-1, 5 days, RNA-seq; Mus musculus; RNA-Seq
RNA was extracted using Direct-zol RNA MicroPrep kit For RNA-seq: Trizol-extracted RNA was proceeded to cDNA generation using the SMARTer V4 Ultra Low RNA Kit (Takara Bio). cDNA was generated with 8 PCR cycles. 1 ng of the resulting cDNA was “tagmented” and barcoded by using the Nextera XT DNA Sample Preparation Kit (Illumina) to generate sequencing libraries for high throughput sequencing. Libraries were purified by AmPure XP magnetic beads (Beckman Coulter), and quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). An equal amount of index libraries were pooled and sequenced (paired-end 50 bp) on an Illumina platform. For ATAC-seq: ATAC-seq was performed using a the method of Corces et al. Three manually dissected cortical collecting ducts (300-600 μm each) were lysed and transposed simultaneously in 25 μl of transposition mix (0.1% NP40, 0.1% Tween-20, 0.01% digitonin and 5% Tn5 enzyme [Illumina # 15027865] in Tagment DNA Buffer [FC-121–1030; Illumina]). The transposition reaction was incubated at 37 °C for 30 mins in a thermomixer with shaking at 1,000 rpm. After 11 tagmentation, the reactions were stopped with addition of 80 μl of water, 3 μl of 0.5% EDTA, 1.5 μl of 20% SDS and 5 μl of proteinase K (20 mg/mL stock, Fisher Scientific #25530049). The transposed DNA fragments were purified by DNA Purification Kits (Zymo Research, # D4014), and amplified by PCR. The final libraries were purified using AmPure XP magnetic beads (Beckman Coulter, Indianapolis, IN). Mitochondrial DNA were depleted from the purified libraries using a CRISPR/Cas9-based method.46 The DNA content of the final library was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). Purified DNA libraries were sequenced on an Illumina Novaseq platform.