Dsdna hs assay

RNA was extracted using the QIAGEN RNeasy kit including the optional on-column DNase treatment. Libraries were prepped using QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) as previously described (Smith et al, 2022 (link)). Libraries were then analyzed for quality using the Agilent High Sensitivity DNA kit and for quantity using Qubit dsDNA HS Assay. Libraries were then pooled and sequenced using NovaSeq 6000 SP Reagent Kit (100 cycles). Libraries were pooled and diluted to 1.25 nM, denatured with 1 M NaOH added to a 0.2 M final concentration (8 min at RT), and quenched with 400 mM Tris–HCl (pH 8). 5% PhiX spike-in (Illumina) was included. Pooled, denatured libraries were run on an Illumina NovaSeq using 76-bp reads, 12-bp index reads, and single-end single-read parameters. Bcl files were converted to FASTQ using bcl2fastq. Sequence quality was confirmed using FastQC v0.11.2. Trimming was performed using BBduk (BBTools v25.92). Alignment used STAR v2.6.0 and reference genomes hg38 and mm9. Differential expression analyses used EdgeR v3.26.8. The analysis in Fig 3E used data originally published previously (Amici et al, 2022 (link)), which were reanalyzed as discussed in the main text. Differentially expressed genes were only considered with adjusted P-values < 0.05.

A total of 250 ng of the same total RNA used for mim-tRNAseq library preparation was used for mRNA-Seq library construction with a Zymo-Seq RiboFree total RNA library kit (Zymo Research, R3000). The libraries were quantified using a Qubit dsDNA HS assay, fragment size was determined on an Agilent TapeStation and the libraries were sequenced for 120 cycles on an Illumina NovaSeq platform, generating >21 × 10⁶ reads per library.

DNA was sheared down to 200 base pairs (bp) using Adaptive Focused Acoustics on the Covaris E220 (Covaris Inc) following manufacturer recommendations with 10 μL Low EDTA TE buffer supplemented with 5 μL of truSHEAR buffer using a microTUBE-15. Libraries were prepared using the Accel-NGS 2 S PCR-Free DNA Library Kit (Swift Biosciences). Ligated and purified libraries were amplified using KAPA HiFi HotStart Real-time PCR 2X Master Mix (KAPA Biosystems). Samples were amplified with 5 μL of KAPA P5 and KAPA P7 primers. The reactions were denatured for 45 seconds (sec) at 98 °C and amplified 13–15 cycles for 15 sec at 98 °C, for 30 sec at 65 °C, and for 30 sec at 72 °C, followed by final extension for 1 min at 72 °C. Samples were amplified until they reached Fluorescent Standard 3, cycles being dependent on input DNA quantity and quality. PCR reactions were then purified using 1x AMPure XP bead clean-up and eluted into 20 μL of nuclease-free water. The resulting libraries were analyzed using the Agilent 4200 Tapestation (D1000 ScreenTape) and quantified by fluorescence (Qubit dsDNA HS assay).