16S-Seq of T-shirt:Human worn
Male subject wore polyester T-shirt 72 h,  10x5 cm section (left axillary region) collected in 50 mL centrifuge tubes.  Textile samples were finely cut onto a sterile petri-dish and weight  adjustedwoth nuclease free H2O. Each sample was divided into 5 lysing tubes  (Fisherbrand Bead Mill Homogenizer Accessory, 2 mL Tough Micro-Organism  Lysing Mix Tubes) with 10 stainless steel beads (2.5 mm) and 10 borosilicate beads (2 mm). DNA  extracted using NucleoSpin Genomic DNA from Soil extraction kit  (Macherey-Nagel), using 420 L of lysis buffer (SL1) and 90 L enhancer  solution (SX) for each tube, thoroughly vortexed, and processed for 2x40 s at  6 m/s using a FastPrep-24 Classic Instrument (MP Biomedical). The lysate was  extracted using a pipette and transferred to Eppendorf tubes. 150 L  precipitation buffer (SL3) was added, the tubes were placed on ice for 5 min,  then centrifuged (11 G, 1 min). Supernatant from the 5 tubes was collected  loaded into 2 NucleoSpin Inhibitor Removal Columns and centrifuged (11 G, 1  min). The remaining extraction was performed according to the manufacturers  guidelines, with elution using 35 L elution buffer. DNA was measured using  Qubit dsDNA HS Assay Kit and stored in the freezer at -18C. Sequencing  libraries were prepared using a dual-PCR setup, targeting variable regions V3  and V4 of the 16S rRNA gene, approx. 460bp. In the first step (30 cycles)  general primers (Uni341F: 5-CCTAYGGGRBGCASCAG-3 and Uni806R:  5-GGACTACNNGGGTATCTAAT-3 were used, and in the second step (15 cycles) the  primers additionally included sequencing adaptors and barcode tags. PCR  reactions were performed using PCRBIO HiFi polymerase, modified to 25 l  reactions (with 2 L template), following the manufacturers instructions.  Amplicon products were purified using HighPrep PCR (AC-60500, MagBio  Genomics Inc., USA) PCR Clean Up System (0.65 L beads:1 L amplicon  product). Samples were normalized using SequalPrep Normalization Plate (96)  Kit (Invitrogen, Maryland, MD, USA), pooled, and then concentrated using the  DNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA, USA). The  pooled library concentration was determined using the Qubit dsDNA HS Assay  Kit (Invitrogen, Thermo Scientific) and adjusted to 1.65 ng/l (4 nM). Paired  end Amplicon sequencing was performed on the Illumina MiSeq Desktop Sequencer  (Illumina Inc., CA, US) with the denatured libraries adjusted to a final  concentration of 16 pM. For each run, 5.0% PhiX was included as internal control  and the MiSeq Reagent Kits v2 were used (Illumina Inc., CA, US). MiSeq  Controller Software was used for sequence demultiplexing.