2 ml screw cap tube

S. gordonii SecCR1 was grown in 100 ml HTVG to late exponential phase of growth. Cells were harvested by centrifugation (10 000 x g, 10 min) and resuspended in 1 ml of phosphate-buffered saline (PBS, 8.7 mM Na₂HPO₄, 1.5 mM NaH₂PO₄, 1.45 M NaCl, pH 7.2) in a 2 ml screw-cap tube (Sarstedt). Glass beads (0.25 ml, 400 μm, VWR International) were added to the cell suspension, and the cells were broken by homogenization (7 bursts of 45 sec each with 1 min cooling between each burst, setting #4,) using a Fast-Prep homogenizer (Thermo Savant, Model FP120). Following homogenization, the suspension was centrifuged (10 s, 10, 000 x g) to sediment the Glass beads, and the supernatant was saved. PBS (0.25 ml) was added to the Glass beads and pulse-centrifuged as above. The supernatant was pooled with the first supernatant. The supernatant was centrifuged (2,000 x g, 3 min) to remove unbroken cells. One ml of the supernatant was centrifuged at 20,000 x g for 20 min. The supernatant was saved as the cytoplasm-cell membrane fraction. The pellet was resuspended in 0.2 ml PBS as the cell wall fraction. Twenty μl of cytoplasm-cell membrane fraction and 4 μl of cell wall, representing the proportional quantity of cell suspension, were analyzed by western blotting.

Each laboratory obtained one 2 mL screw-cap tube (Sarstedt, Nümbrecht, Germany; labelled: SeqForSTRs, SeqPool 20 μL, ForenSeq Kit, 29.11.2017 PM) containing 20 μL pooled sequencing library. The final library pool was made of 25 selected DNAs, a negative and a positive amplification control. To ensure sufficient library volume OL simultaneously prepared three library pools, including the same samples and index adapters (i5 and i7) using the ForenSeq DNA Signature Prep Kit, primer mix A (Verogen, [22 ]) in one 96-well plate (Table S1). Library preparation, purification and normalization were performed according to [22 ]. All samples were amplified with 1 ng DNA according to [22 ], except for G49-S4 (2.6 ng DNA), the samples for the sensitivity study (serial dilution: 1000 pg–31 pg DNA) and aDNA samples.
Prior to pooling, 30 μL of each library sample (present in pools 1, 2 and 3; Table S1) was joined into a 0.8 mL deep-well storage plate (one sample/well). Libraries were pooled, diluted and denatured following [22 ] before loading into a MiSeq FGx Reagent Cartridge and sequenced on the MiSeq FGx instrument ([32 ], Table S2). To achieve uniform designation for all sequencing runs OL provided a text-based sample sheet including relevant information on, e.g. sample name as well as adapter combination needed for demultiplexing and data analysis (Table S3).

Following collection, blood samples were left to clot overnight at room temperature. Samples were centrifuged for ten minutes at 2000× g. Serum was removed using a sterile pastette into a labelled 2 mL screw cap tube (Sarstedt, Nümbrecht, Germany). All samples, including tissue samples, were stored at −70 °C for future downstream processing and analyses.