Dna rna shield lysis tube

Sufficient DNA was extracted from 296 stool sample aliquots stored in DNA/RNA shield lysis tubes (Zymo Research, Irvine CA) using the ZymoBIOMICS 96 MagBead DNA kit (Zymo Research). Extracted DNA was quantified using QuantIT dsDNA Assay kit, high sensitivity (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. Three (3) samples were omitted from downstream analysis due to failure to meet input requirements for library preparation. Libraries were prepared for each sample using the Illumina Nextera DNA Flex library kit (Illumina, San Diego, CA) with unique dual indexes according to manufacturer guidelines. Libraries were pooled and submitted to UC Davis DNA Technologies Core for sequencing on an Illumina NovaSeq S4 flow cell (Illumina, San Diego, CA). Each lane of the S4 flow cell contained 96 libraries.

Prior to sequencing, isolates were re-cultivated in tryptic soy broth, mixed in DNA/RNA Shield lysis tubes^™ (Zymo Research^™) and centrifuged at 10,000 x g for 1 minute. DNA was isolated using the ZymoBIOMICS DNA isolation kit following the manufacturer’s recommended protocol (Zymo Research^™). More specifically, the resulting supernatant was added to a Zymospin^™ filter, centrifuged at 8000 x g for 1 minute followed by addition of DNA binding buffer. The resulting mixture was added to a Zymospin^™ column, centrifuged at 10,000 x g for 1 minute followed by rinses with DNA wash buffer. This was added to DNAse/RNAse free water and centrifuged at 10,000 x g for 1 minute. DNA was eluted from this using a Zymospin^™ filter via centrifugation. The resulting DNA was prepared for Illumina next-generation sequencing using the Illumina Nextera XT DNA library prep kit, per recommended instructions. Completed sequencing libraries were assessed for quality and concentration by gel electrophoresis (Agilent^™) and Qubit fluorometric quantitation (Thermo Fisher Scientific^™), respectively. Completed libraries were pooled in equimolar ratios and underwent whole genome sequencing via 2x250 bp sequencing using v3 sequencing reagents on an Illumina MiSeq (see S1 Table for number of sequencing reads).

All experiments involving human subjects were carried out according to the IRB protocols approved by UCSD (Project#071032). Adult subjects with moderate to severe atopic dermatitis or psoriasis, and matched healthy subjects were recruited form the UCSD, San Diego. Demographic data of all human studies are provided in Tables S1–S4. To correct live bacteria or microbial DNA, swab was prewet with sterile saline or TE buffer supplemented with 0.05% Tween 20 and 0.1% Triton X-100, respectively. Collection of live bacteria was done by swabbing lesional skin on the antecubital fossa of patients with atopic dermatitis and on elbow or upper arm of patients with psoriasis, or upper arm of healthy subjects (3 cm²) by stroking 20 times with constant pressure. Live bacterial swab was stored immediately in 85% tryptic soy broth and 15% glycerol at −80°C. DNA swab was stored in DNA/RNA shield Lysis tube (ZymoResearch) at −80°C until DNA extraction process.