Nextera index kit

ATAC-seq was performed on FACS-sorted neonatal (PND3) and BAL-isolated adult alveolar macrophages. Fifty thousand cells were washed once in ice-cold PBS by centrifugation at 500g for 5 min at 4 °C. Cells were resuspended in 50 µl lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂ and 0.1% IGEPAL CA-630) and spun down immediately at 500g for 10 min at 4 °C. Pellets were resuspended in transposition reaction mix (25 µl 2× TD Buffer (Illumina, FC-121-1030), 2.5 µl Tn5 Transposes (Illumina, FC-121-1030) and 22.5 µl nuclease-free H₂O) and incubated at 37 °C for 30 min. DNA was purified with a Qiagen MiniElute kit and amplified with Nextera PCR primers (Illumina Nextera Index kit) and NEBNext PCR master mix (M0541, New England BioLabs). Amplified DNA was purified with a Qiagen Mini Elute kit. Libraries were sequenced paired-end on a Novaseq sequencer at the University of Chicago Genomics Facility.

The V3-V4 hypervariable regions of bacterial 16S rRNA gene region were amplified using the following primer set: 341F-CCTACGGGNGGCWGCAG and 785R-GACTACHVGGGTATCTAATCC. The targeted gene region has been shown to be suitable for Illumina sequencing^{118 (link)}. Each library was prepared with a two-step PCR protocol based on Illumina’s “16S metagenomic library prep guide” (15044223 Rev. B), NEBNext® Q5 Hotstart High-Fidelity DNA polymerase (New England BioLabs Inc.) according to the manufacturer’s protocol using Q5® Hot Start High-Fidelity 2X Master Mix (NEBNext - New England BioLabs, PCR under the following conditions: 98 °C for 30 sec for initial denaturation of the DNA, followed by 25 cycles of 98 °C for 10 sec, 55 °C for 30 sec, 72 °C for 20 sec and additionally 72 °C for 2 min), and the Nextera Index kit (2 × 250 bp). Paired-end (PE, 2 × 250 nt) sequencing with a 5% PhiX spike-in was performed with an Illumina MiSeq (MiSeq Reagent kit v2) at Genomed, Warsaw, Poland; following the manufacturer’s run protocols (Illumina, Inc., San Diego, CA, USA). The automatic primary analysis and the de-multiplexing of the raw reads were performed on the MiSeq machine, with the use of MiSeq Reporter (MSR) v2.6 (16S Metagenomics Protocol).

The V3–V4 hypervariable regions of bacterial 16S rDNA region were amplified using the following primer set: 341F - CCTACGGGNGGCWGCAG and 785R - GACTACHVGGGTATCTAATCC. The targeted gene region has been shown to be the most appropriate for the Illumina sequencing (Klindworth et al., 2013 (link)). Each library was prepared in a two-step PCR protocol based on Illumina's “16S metagenomic library prep guide” (15044223 Rev. B) using NEBNext® High-Fidelity 2xPCR Master Mix (New England BioLabs) and Nextera Index kit (2 × 250 bp). Paired-end (PE, 2 × 250 nt) sequencing, with a 5% PhiX spike-in, and was performed on an Illumina MiSeq (MiSeq Reagent kit v2) following manufacturer's run protocols (Illumina, Inc., San Diego, CA, USA), at Genomed, Warsaw, Poland. The automatic primary analysis and the de-multiplexing of the raw reads were performed on MiSeq with the use of MiSeq Reporter (MSR) v2.4 (BaseSpace).