title: GSM5191745: Adrenal_Glands_WT_3; Mus musculus; RNA-Seq
library_construction_protocol: KO and WT adrenal glands were homogenized, and their total RNA was extracted by RNeasy kit (Qiagen). The total RNA was quantified using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Inc.), and its integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies). rRNA was depleted from 250 ng of total RNA using QIAseq FastSelect (Human 96rxns). cDNA synthesis was achieved with the NEBNext RNA First-Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules (New England BioLabs). The remaining steps of library preparation were performed using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), according to the manufacturer's instructions. Adapters and PCR primers were purchased from New England BioLabs. Libraries were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). The average fragment size was determined using a LabChip GX (PerkinElmer). The libraries were normalized and pooled and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 225 pM on an Illumina NovaSeq S2 lane using the Xp protocol as per the manufacturer's recommendations. The run was performed for 2 x 100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at a 1% level. Base-calling was performed with RTA v3. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads.