Nextseq 2000 sequencing platform

BMDNs treated with PBS or eCIRP (1 g/mL) for 2 hours were sorted for scRNA-Seq using the 10× Genomics Chromium platform (41 (link)). Library preparation was conducted according to the recommended protocol for the NextGEM Single-Cell 3’ Library Kit v3.1 (no. 1000121; 10× Genomics). Libraries were sequenced on the Illumina NextSeq2000 sequencing platform to a mean depth of approximately 40,000 reads per cell. The Cell Ranger count pipeline (v6.0.0, 10× Genomics) was used to align FASTQs to the mouse reference genome (gex-mm10-2020-A, 10× Genomics) and produce digital gene-cell counts matrices and to perform quality control of the mapping results. Primary assessment with Cell Ranger for the PBS-treated sample reported 6,104 cells with a median of 4,624 unique molecular identifiers per cell and median of 1,284 genes per cell at 64.8% sequence saturation, with a median of 37,255 reads per cell. Primary assessment with Cell Ranger for the eCIRP-treated sample reported 5,711 cells with 4,460 unique molecular identifiers per cell and 1,250 median genes per cell at 70.0% sequence saturation, with a median of 44,261 reads per cell.

DNA was extracted from fecal samples using QIAamp PowerFecal Pro DNA kits (QIAGEN). Samples were thawed and ∼250 mg of fecal sample was lysed via bead beating. According to the kit protocol, the sample was then cleaned of non-DNA organic and inorganic material, and then washed using ethanol. DNA was eluted into 10 mM Tris and quantified using the Qubit 1X dsDNA High-Sensitivity (HS) Assay Kit (Thermo Fisher Scientific) using the Qubit fluorometer (Thermo Fisher Scientific). DNA at a concentration of 10 mg/µl was sent for sequencing. Samples were sent in bulk for sequencing blinded to sample timepoints of each patient. Shotgun metagenomic sequencing was performed with Illumina NextSeq 2000 sequencing platform using paired-end reads of 150 bp in length, resulting in a median of ∼28 million reads per sample.

Individual serum samples were pre-treated with Turbo DNAse (ThermoFisher Scientific (Waltham, MA, USA)) for host/bacterial DNA removal and were further pooled. The pooling of samples is a standard procedure in the metagenomic analysis in order to reduce the cost of the sequencing and to test the largest amount of samples possible. The whole pool volume was extracted using the High Pure Viral Nucleic Acid Large Volume kit (Roche, Basel, Switzerland). The extracted nucleic acids were submitted to reverse transcription using the Superscript III First-Strand Synthesis System (ThermoFisher Scientific) and amplification was performed by the QuantiTect Whole Transcriptome Kit (QIAGEN, Hilden, Germany). The libraries were prepared using the DNA Prep Library Preparation Kit (Illumina (San Diego, CA, USA)). The pair-end sequencing of the dual-indexed libraries was performed by Illumina NextSeq 2000 sequencing platform using the NextSeq P3 flowcell (300 cycles) (Illumina), following the manufacturer’s instructions.