Dsdna hs assay

Nα and Nβ neutrophils from peritoneal washes of infected mice were FACS sorted based on their expression of CD11b and Ly6G and FSC/SSC characteristics (Supplementary Fig 1a). Up to 1000 cells of each subtype were directly sorted in a 96-well plate containing Lysis Buffer and RNase Inhibitor. One sample was excluded for technical reason. cDNA amplification was performed directly from lysed cells, with no previous RNA isolation, using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara). Amplified cDNA was used for preparing index tagged sequencing libraries using the Nextera XT kit (Illumina). Average library size and size distribution were determined using the 2100 Bioanalyzer High Sensitivity DNA chip (Agilent). Libraries were quantified with the Qubit fluorometer using the dsDNA HS assay (ThermoFisher Scientific). Libraries were normalized and sequenced as a pool in one lane of a Rapid flow cell in the HiSeq2500 sequencer (Illumina) to generate 61 bases single-end reads. FastQ files were obtained using the bcl2fastq v2.20.0.422 software (Illumina).

Shell samples were broken into smaller pieces using a mortar and pestle, re-weighed, and placed in a SafeLock two mL tube (Eppendorf) with a stainless steel five mm bead (Qiagen). All samples and two blank tubes (steal bead but no shell) were processed using a TissueLyser II machine (Qiagen) at the maximum frequency setting (30 Hz) for five minutes. DNA was extracted following a salting-out method (Gemmell & Akayama, 1996 (link)), with these modifications: (1) lysis incubation was performed overnight; (2) the resulting DNA was resuspended in 100 µL low EDTA TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8). DNA yields were determined using a Qubit^® 2.0 Fluorometer (ThermoFisher Scientific), using the dsDNA HS Assay (ThermoFisher Scientific). DNA (ng)/shell (mg) sample ratios were calculated for each sample. Shell valves from beach-stranded specimens were very brittle, and were processed solely with mortar and pestle. DNA from these samples was eluted in 40 µL low EDTA TE buffer.

The concentration of the isolated cfDNA was analyzed in duplicate by fluorometric quantification using the Qubit Fluorometer 3.0 (Thermo Fisher, Waltham, MA, USA) and the dsDNA HS Assay (quantification range: 10 pg/µL–100 ng/µL; Thermo Fisher). For four of the donors, we determined the representative quantity and fragment size profiles of cfDNA using a 2100 Bioanalyzer (Agilent Inc., Santa Clara, CA, USA) with the Agilent High-Sensitivity DNA Kit.

We used single specimen samples from four eusocial species (S. chacei, S. filidigitus, S. microneptunus, and S. regalis) and four noneusocial species (S. carpenteri, S. hoetjesi, S. kensleyi, and S. pandionis) for DNA extraction and low-coverage whole-genome sequencing. These species included three independent origins of eusociality, as well as two species from each of the four Synalpheus major clades (supplementary table S1, Supplementary Material online). The field collection protocol has been reported in Macdonald et al. (2006) . We extracted genomic DNA using several walking legs from alcohol-preserved specimens with Qiagen DNeasy Tissue Kits (Qiagen). Extracted DNA was quantified using a Qubit 3.0 Fluorometer with the dsDNA HS assay (ThermoFisher Scientific) and visualized on 2% agarose gels. We provided 1,500 ng of genomic DNA to Novogene (Chula Vista, CA) for TruSeq PCR-free library preparation (Illumina) and 150-bp pair-end sequencing on an Illumina NovaSeq to obtain at least 1× coverage according to published genome size (4.8–11.8 GB) (Jeffery et al. 2016 (link)). Low-coverage whole-genome sequencing reads from whole-cell extraction contain a high copy number of extranuclear sequences, and it has been shown to be an efficient and economical approach to assemble complete mitochondrial genomes (Dierckxsens et al. 2016 ).

DNA was extracted from blood using the QIAcube system with QIAmp DNA kit (Qiagen, Hilden, Germany), and 10 ng were used for sequencing by NGS technology. The customized designed SNP panel was composed of n =139 SNPs (Supplementary Table S1), present in n =89 amplicons (size range 125–375 bp). This panel permitted to identify 417 genetic variants in total (Supplementary Table S1). The analysed SNPs were selected based on an extensive review of articles on the association between psoriasis and SNPs or response to biologics [7 (link),9 (link),12 (link),18 (link),22 (link),23 (link),24 (link)].
NGS was performed using the Ion GeneStudio™ S5 Plus platform (Thermo Fisher Scientific, Massachusetts, USA). Libraries were amplified by the Ion AmpliSeq™ Library kit Plus (Thermo Fisher Scientific) and quantified using the Qubit 4 Fluorometer and 2100 Bioanalyzer with dsDNA HS assay and High Sensitivity DNA kit (Thermo Fisher Scientific), respectively. Sequencing data were processed with the Ion Torrent Suite software v.5.10.
Positive calls were selected with a read depth >30X and allelic frequency higher than 0.3 (range 0–1.0). Reads were aligned to human genome sequence (build GRCh37/human genome 19). Variants were collected using Variant Caller plugin and systematically evaluated, filtered, and annotated using tailored R scripts. Variants’ annotations were finally verified using ANNOVAR.

Leftover peripheral blood samples after routine clinical tests were collected from patients with ovarian cancer (n = 289) or pancreatic cancer (n = 4) in Shanghai General Hospital. Genomic DNA (gDNA) was extracted from blood using the QIAamp Blood Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration of genomic DNA was measured by Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA) using dsDNA HS assay (Q32854). The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai General Hospital (No. 2022KY019). Requirement for informed consent was waived by the ethics committee.