RNA-Seq of Mus musculus: K4APE Male TechRep1
RNA extraction was performed using the PureLink RNA Mini Kit (12183018A, Thermo Fisher Scientific) according to the manufacturer's guidelines. Briefly, cells were trypsinized, pelleted, lysed, and homogenized with a 21-gauge needle. Afterwards, 70% ethanol was added to the cell lysates, and the mixtures were then loaded onto spin cartridges to bind the RNA. Following three washes, RNA was eluted in RNase-free water. The quality of the extracted RNA was assessed using a Bioanalyzer 2100, and quantification was done using a Qubit fluorometer (Q10210, Invitrogen). For the preparation of Illumina library, strand-specific RNA-sequencing (ssRNA-seq) libraries were generated using the Illumina Truseq Stranded mRNA Sample Preparation Kit (RS1222101, Illumina), adhering to the manufacturer's guidelines. Succinctly, 1 g of total RNA was used to purify mRNA via oligo(dT) coupled to magnetic beads. The mRNA was then fragmented, and cDNA was synthesized utilizing SuperScript III Reverse Transcriptase (18080044, Thermo Fisher Scientific). This was followed by adenylation at the 3 end, barcoding, and adapter ligation. The adapter-ligated cDNA fragments were subsequently enriched and purified with Agencourt Ampure XP beads (A63880, Agencourt). The integrity of the libraries was validated using the DNA 1000 kit on a 2100 Bioanalyzer (50671504, Agilent Technologies) and quantified with a Qubit fluorometer (Q32850, Life Technologies). The ssRNA libraries underwent sequencing on the Illumina HiSeq4000 platforms. Sequenced reads were then mapped to the Mus musculus genome GRCm39 (GENCODE Release M33). For RT-PCR, total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems). The reaction mixture contained 1 g of total RNA, 2 l of RT buffer, 0.8 l of 100 mM dNTP mix, 2 l of 10x random primers, 1 l of MultiScribe Reverse Transcriptase, and nuclease-free water bringing the total volume to 20 l. The mixture was incubated for 10 minutes at 25C, followed by 2 hours at 37C. Enzyme inactivation was achieved by heating the mixture to 85C for 5 minutes. For the real-time PCR, a 10 l reaction mix was prepared containing 20-30 ng cDNA template, 5 l of KAPA SYBR FAST Universal 2X qPCR Master Mix (KK4602, Sopachem Life Sciences), and 500 nM each of forward and reverse primers (Integrated DNA Technologies)designed to amplify target genes. The PCR reaction was carried out on using the CFX96 Real-Time System thermocycler (C1000, Biorad). GAPDH and -actin served as reference genes. Results were analyzed using the 2CT method, with error bars illustrating the standard error of the mean (SEM) from three biological replicates.
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K4APE-MiceF3681-TechRep1