Random primer 9

Purified DNA underwent second-strand synthesis using Sequenase Version 2.0 (Thermo Fisher, UK) and random primer 9 (New England BioLabs, UK). Libraries were prepared from 1 ng of the resulting double-stranded DNA using the Nextera XT kit (Illumina, UK) and sequenced on the MiSeq platform with reagent kit v2 (500 cycles). After adapter removal and quality trimming using Trimmomatic [36 (link)], reads were aligned against the reference sequence (GenBank no. J02275) and the derived mutant sequences using Bowtie 2 [37 (link)].

Purified RNA was reverse transcribed using Random Primer 9 (NEB) and SuperScript II reverse transcriptase under error prone conditions (Smola et al., 2015 (link)). The resultant cDNA was purified using G50 column (GE Healthcare) and subjected to second-strand synthesis (NEBNext Second Strand Synthesis Module). A standard Nextera DNA library protocol (Illumina) was used to fragment the cDNA and add sequencing barcodes. Samples were purified using Ampure XP beads (Beckman Coulter), and quantification of the libraries was performed with Qubit dsDNA HS Assay kit (ThermoFisher Scientific). Final libraries were run on Agilent Bioanalyzer for quality check. Gel purification (GeneJET, ThermoFisher Scientific) was performed as needed to remove primer dimer bands from libraries before sequencing. Libraries were sequenced as paired-end 2 × 151 read multiplex runs on MiSeq platform (Illumina). Sequenced reads have been uploaded to the NCBI SRA database under BioProject ID PRJNA762079 for in-cell data and PRJNA812003 for cell-free data.

Two hundred and fifty microliters of homogenized tissue were suspended in 1 ml of Trizol (ThermoFisher) and frozen at −80°C until extraction. RNA was extracted following manufacturer’s protocol. Extracted RNA was DNase treated with 10 units RQ-1 RNase free DNase (Promega) following manufacturer’s protocol. RNA was purified after DNase treatment using RNA Clean and Concentrator-25 (Zymo) and eluted in 30 μl of H2O, and quantified using a NanoDrop spectrophotometer. Five hundred nanograms of total RNA were used to generate cDNA using SuperScript II Reverse Transcriptase (ThermoFisher) and Random Primer 9 (NEB) following manufacturer’s protocol. Copy number of lasA and gyrA were quantified using iTaq Universal Sybr Green master mix (Bio-Rad) in 20 ul reaction volumes on a Roche LightCycler 96, using the following primer pairs: lasA_RT_5 5’-CCTGTTCCTCTACGGTCGCG-3’, lasA_RT_3 5’-GGTTGATGCTGTAGTAGCCG-3’, gyrA_5_RT 5’-GAAGCTGCTCTCCGAATACC-3’, gyrA_3_RT 5’-CAGTTCCTCACGGATCACCT-3’.

First stand cDNA synthesis of treated RNA was carried out as previously described (22 (link)). Briefly, 29 μl of purified RNA was added to 100 ng Random Primer 9 (NEB), and 0.2 mM of each dNTP. This mix was then incubated at 65°C for 5 min before being transferred to ice, followed by addition of 50 mM Tris (pH 8.0), 75 mM KCl, 10 mM DTT, and 6 mM MnCl₂ and 2 μl of SuperScript II Reverse Transcriptase (Invitrogen), supplemented with 20 U of murine RNase inhibitor (NEB) to achieve a final volume of 40 μl. The reaction was incubated at 25°C for 10 min, then 42°C for 3 h, then 70°C for 15 min. First strand cDNA was desalted using G50 columns (GE). The desalted product was then introduced into a second strand synthesis reaction. The cDNA second strand was generated using mRNA Non-Directional Second Strand Synthesis module (NEB). Double stranded DNA was then cleaned up using Ampure XP purification beads and eluted in 30 ul of water and quantified using a Qubit dsDNA HS Assay (Invitrogen). Double-stranded DNA was fragmented, repaired, and adaptor ligated for sequencing library preparation using the NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina^®. Library quality was assessed using the High Sensitivity Bioanalyzer protocol (Agilent). Libraries were then loaded on an Illumina MiSeq at 10 pM and run at 300 cycles (150 × 2) or 600 cycles (300 × 2).

BMDMs were isolated as described above and differentiated for 7 days, at which point their RNA was isolated using the RNeasy mini kit from Qiagen.
Total RNA was reverse-transcribed using RNasin Plus (Promega), 2.5 mM dNTPs, Random Primer 9, and M-MuLV Reverse Transcriptase from NEB. ADAM17 oligonucleotides were purchased from Eurofins Genomics using previously described sequences (72 (link)), ADAM17 Forward 5’-GATGCTGAAGATGACACTGTG-3’ (A17 exon 14); ADAM17 Reverse 5’- GAGTTGTCAGTGTCAACGC-3’ (ADAM17 exon 14–15). GAPDH oligonucleotides were purchased from Qiagen. ADAM17 mRNA in wild-type and Adam17Δcyto tissues was quantified via RT-qPCR using SYBR Green Reagent and an ABI PRISM 7900HT cycler (both from Applied Biosystems, Thermo Fisher Scientific). GAPDH was used as a housekeeping control. Three independent experiments were performed with duplicate or triplicate samples for each experiment.