Dneasy 96 powersoil pro qiacube ht kit

DNA was extracted from stool samples using a modified DNeasy 96 PowerSoil Pro QIAcube HT kit (QIAGEN, Copenhagen, Denmark). Stool samples were placed on dry ice where a 0.5 g subsample of frozen stool was transferred into a sterile 2 mL cryotube under aseptic conditions in a biosafety cabinet using 4 mm sterile punch biopsies with plunger (Scandidact, Odder, Denmark) and were subsequently stored at −80 °C until DNA extraction. DNA was extracted using the high-through put extraction method by Jensen et al. [25 ]. In short, samples were thawed on ice, and 100 µL of stool (∼125 mg) from each sample was transferred to a 1.2 mL matrix tube prefilled with Lysing Matrix E (MP Biomedicals, Santa Ana, CA). Then, 500 µL of CD1 was added to each tube, and samples were bead-beaten as follows: three cycles of 1600 rpm for 120 s with two minutes incubation on ice between each cycle (FastPrep-96™) followed by 10 min of centrifugation (3486 × g). Afterwards 300 µL of supernatant was transferred to an S-block containing 300 µL of CD2 solution and 100 µL of nuclease-free water per well. The remaining steps were performed using the QIAcube HT kit (QIAGEN, Copenhagen, Denmark). DNA concentrations were measured using a Qubit dsDNA HS kit (Thermo Fisher Scientific, Waltham, MA) on an Infinite 200 Pro [25 ].

Self-collected combined nose-throat swabs were stored at −20°C at the participants’ houses until the end of the study. After transportation on ice, samples for DNA analysis were kept at −20°C in the laboratory until further processing. Prior to DNA extraction, all samples were vortexed for 15 to 30 s, and 500 μL of the eNAT buffer was used for automatic extraction using a DNeasy 96 PowerSoil Pro QIAcube HT kit (Qiagen). Negative extraction controls were included at regular time points throughout the study. All samples were eluted with 100 μL elution buffer, and DNA concentrations were measured using the Qubit 3.0 fluorometer (Life Technologies, Ledeberg, Belgium).
Amplicon sequencing (V4 region of the 16S rRNA gene) was performed using an in-house-optimized protocol (48 (link), 49 (link)). Processing and quality control of the reads were performed using the R package DADA2, version 1.6.0, to achieve amplicon sequence variant (ASV)-level counts. Taxonomic annotation was performed using the ezbio 16S database (retrieved June 2018). All data handling and visualization were performed in R, version 3.4.4, using the tidyverse set of packages and the in-house package tidyamplicons, version 0.2.1 (publicly available at https://github.com/SWittouck/tidyamplicons), as described previously (48 (link)).

For gut microbiome analysis, we aimed to collect the first stool sample as early as possible following a patient’s admission to the ICU, ideally within 72 h of the first kefir dose. This timing was contingent on whether the patient had a bowel movement within this period. Considering the initial dosing regimen of kefir, its impact on the gut microbiome at this early stage was anticipated to be minimal. The second stool sample was planned for collection after at least 72 h of administering the full dose of kefir to the patients.
Following study completion, all stool samples were sent to the University of Minnesota Genomics Center for DNA extraction and sequencing. Fecal DNA was extracted using Qiagen’s DNeasy 96 PowerSoil Pro QIAcube HT Kit following the manufacturer’s instructions (QIAGEN, Germantown, MD, USA) and was quantified using a NanoDrop-8000 UV-Vis Spectrophotometer (ThermoScientific, Wilmington, DE, USA) and PicoGreen assays. Samples were then loaded onto the QIAcube HT, an automated DNA extraction instrument. DNA was quantified using Qubit before preparing sequencing libraries using the Nextera XT protocol. Metagenomic sequencing libraries were loaded onto an Illumina Novaseq 6000 sequencer or smaller-scale Illumina sequencing instrument using the same 2 × 150 bp chemistries, targeting 8 M paired-end reads per sample.

Individual mice were allowed to defecate normally in autoclaved cages with no bedding. Using a sterile 26Gx1/2 needle (one per mouse), the first two fecal pellets per mouse were submerged in stabilization buffer in a barcoded sample collection tube. Collection tubes were shipped to TransnetYX for DNA extraction (Qiagen DNeasy 96 PowerSoil Pro QIAcube HT kit/protocol), library preparation (KAPA HyperPlus protocol), and sequencing. High molecular weight genomic DNA captured the true microbial diversity of stool samples from two cohorts: six- to eight-week-old Hltf^+/+ (n = 9) and Hltf^-/- (n = 5) immune deficient (ID) male mice. Libraries were sequenced using Illumina NovaSeq at a dept of 2 million read pairs (2x150 configuration) sufficient for species and strain level taxonomic resolution. FASTQ files were uploaded onto One Codex analysis software [11 (link)] and analyzed against the One Codex database of ~127K whole microbial reference genomes. Classification results were filtered through several statistical post-processing steps to eliminate false positive results caused by contamination or sequencing artifacts. Mouse 12K metagenome-assembled genomes were included to screen out host reads. One Codex annotated metagenomes and generated a taxa plot of the top 10 species for all samples, and calculated alpha (within-sample) family diversity for comparison of the two cohorts.

Faecal samples were extracted on the QIAcube HT system using the DNeasy 96 PowerSoil Pro QIAcube HT Kit (Qiagen 47021, Hilden, Germany), according to the manufacturer’s instructions, with a modified initial processing step (Qiagen 9001793). Mechanical lysis was performed using PowerBead Pro beads (Qiagen 19311). The resulting DNA was quantified using QuantIT, a high-sensitivity dsDNA fluorometric assay (Thermo Fisher, Q33120, Waltham, MA, USA). Samples were required to reach a minimum of 0.2 ng/μL to pass quality control requirements.