Nextseq 2000

HyDrop-ATAC libraries were sequenced on Illumina NextSeq500 or NextSeq2000 systems using 50 cycles for read 1 (ATAC paired-end mate 1), 52 cycles for index 1 (barcode), 10 cycles for index 2 (sample index), and 50 cycles for read 2 (ATAC paired-end mate 2).
HyDrop-RNA libraries were sequenced on Illumina NextSeq2000 systems using 50 cycles for read 1 (3-prime cDNA), 10 cycles for index 1 (sample index, custom i7 read primer), 10 cycles for index 2 (sample index), and 58 cycles for read 2 (barcode+ UMI, custom read two primer).

Approximately 200mg of stool was weighed out and diluted to 1:6 with SM Buffer then was vortexed at max speed for three minutes to fully homogenize. Samples were centrifuged at 4° for five minutes at 20,000g. The centrifuged pellet (metagenomics) was taken through Qiagen’s DNeasy PowerSoil Pro Kit following kit protocol recommendations and then followed by Illumina’s DNA prep for library builds and sequenced on a NextSeq 2000 (2×150). The supernatant (viral) was filtered by a 0.20μm filter and then processed by a VLP enrichment before extractions. A mastermix of Benzonase (2μl), Baseline Zero 10x Buffer (100μ), and Baseline Zero DNASE (4μl) was added to each sample and heated for 37° for one hour. After the heat treatment 1mL was transferred into bioMérieux’s EMAG for total nucleic acid (TNA) extraction. The TNA was amplified with GenomiPhi V2 (GE Healthcare) before moving into library builds with DNA prep (Illumina) and sequencing with a NextSeq 2000 (2×150). Controls of SM Buffer spiked with lambdavirus DNA were used to assess cross-sample contamination during amplification and sequencing.

Crosslinked ChIP–seq was performed like crosslinked SCAR-seq described above, except for omitting EdU labeling and all associated steps for isolation of labeled DNA strands (that is, Click-iT, streptavidin capture and strand separation). Further adaptations of the protocol are described here. ChIP reactions were set up in 1.5-ml tubes with 50 μg of mouse chromatin and 1 μg of Drosophila chromatin (2% spike-in) and incubated with anti-SUZ12 antibody in a total volume of 1 ml (Supplementary Table 2 for antibodies). Two reactions were set up per cell line for a total of 100 μg starting material, and samples were combined after ChIP washes and decrosslinking during purification with a MinElute Reaction Cleanup kit (Qiagen, 28204) by loading the same column twice. A total of 30 μl of Invitrogen Dynabeads Protein G were added per 50 μg of chromatin. Libraries were amplified in seven PCR cycles. ChIP inputs were prepared for all samples and replicates in parallel.
Samples were sequenced on a NextSeq500 or NextSeq 2000 (Illumina). All samples are detailed in Supplementary Table 1. ChIP–seq data processing and analysis are detailed in the Supplementary Methods.

Total DNA of erythromycin-resistant colonies was purified using the DNeasy Blood & Tissue Kits (Qiagen). Genomic DNA library preparation and sequencing were performed at the SeqCenter (Pittsburgh, PA) by using Illumina NextSeq 2000 and NovaSeq 6000 technology. Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10 bp unique dual indices (UDI). Demultiplexing, quality control and adapter trimming were performed with BCL Convert (Version 4.1.5). To analyze genetic alterations by HGT, paired-end sequencing reads (2 × 151 bp) were assembled by SPAdes (Version 3.15.2)^{62 (link)}. The sequencing reads (2 × 151 bp) were also mapped to the plasmid bearing the thyA gene (shown in Fig. 4a) and the plasmid with transgenes (shown in Fig. 5a), using Bowtie 2 (Version 2.4.5)^{63 (link)}. Raw sequencing data and assembled genomes have been deposited at DDBJ/ENA/GenBank under the accession number: PRJNA754595.