GSM7854781: HSV-ZFP189-WT, 24h, Bio Rep 4; Mus musculus; RNA-Seq
Total RNA was extracted from fresh frozen tissue samples using Qiagen RNeasy Plus Universal mini kit following manufacturer's instructions (Qiagen, Hilden, Germany). Samples were initially treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contaminants. The next steps included performing rRNA depletion using QIAseq® FastSelectTM−rRNA HMR kit (Qiagen, Germantown, MD, USA), which was conducted following the manufacturer's protocol. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations. Briefly, enriched RNAs are fragmented for 15 minutes at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3'ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration.