Ampure bead

Stool samples were collected from mice before, 2 weeks after, or 4 weeks after antibiotic treatment with CPFX, IPM, MDZ, and VCM and frozen and stored at −80 °C. DNA extraction was done as described previously with some modifications71 (link). The stool samples were homogenized in TE buffer and treated with Achromopeptidase and Lysozyme at 37˚C for an hour. DNA was purified with phenol/chloroform extraction and isopropanol precipitation. 16S rRNA gene fragment including V3 and V4 region were amplified by PCR (forward primer: ACACGACGCTCTTCCGATCTCCTACGGGNGGCWGCAG, reverse primer: GACGTGTGCTCTTCCGATCTGACTACHVGGGTATCTAATCC, underline: overhang sequence for 2nd PCR) for 20 cycles, and PCR products were purified with Agencourt AMpure beads (Beckman coulter). Then, overhang and index sequences for sequencing were added by 2nd PCR with NEBNext multiplex Oligos for Illumina (Dual Index Primers Set1, New England Biolabs) for 8 cycles and the products were purified with Agencourt AMpure beads.
16S V3-V4 rDNA in the stool DNA sample, normalized with stool amount (40 μg), were amplified by PCR with the 1st primers for 24 cycles. Then agarose gel electrophoresis was performed and 16S rDNA product bands were semi-quantified by ImageJ (NIH software).

MeDIP and hMeDIP were performed as previously described^{35 (link)}. For methylated and hydroxymethylated DNA immunoprecipitation (IP), genomic DNA was extracted using phenol-chloroform. One microgram of genomic DNA was used per IP with similar procedure as ChIP described previouslys. Purified genomic DNA was fragmented by Covaris (Covaris) and mixed with 1 µg of 5mC (eurogentec) and 5hmC (Active motif) antibody conjugated with Dynabeads®M-280 Sheep Anti-Rabbit IgG (Invitrogen) respectively. DNA fragments pulled down from MeDIP and hMeDIP, as well as genomic DNA (input) were end-repaired by T4 DNA polymerase and phosphorylated. A single ‘A’ base was added to the 3′ end with Klenow. Adaptors with indexes were ligated to the fragments with multiplexing sample preparation kit (Illumina). Ligation products between 300 and 500 bp were purified using AMPure beads (NEB) and amplified by PCR. Libraries were quantified with PicoGreen and QC with Bioanalyzer then analysed by Illumina Hiseq2000 platform. The experiments were performed three times.

Primary GB whole-genome bisulphite library preparation was carried out as described previously^{20 (link)}. Briefly, 5 μg of genomic DNA was sheared using a Covaris device. After adaptor ligation, DNA fragments with insert lengths of 200–250 bp were isolated using an E-Gel electrophoresis system (Life Technologies) and bisulphite converted overnight using the EZ DNA Methylation kit (Zymo Research). The fragments were PCR amplified using the FastStart High Fidelity PCR kit (Roche) for 6–8 cycles. Library aliquots were then purified and size selected with AMPure beads (New England BioLabs) and quality controlled with a Bioanalyzer (Agilent). Each library was sequenced using 2 lanes on an Illumina HiSeq 2000 in the DKFZ Genomics and Proteomics core facility.

mRNA library was prepared using NEBNext Utra mRNA Library Prep Kit for Illumina (NEB) following manufacturer’s protocol. Briefly, total RNA was extracted following manufacturer’s instructions (TRIzol, Invitrogen). mRNA was isolated using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB) and fragmented using Covaris (Covaris). cDNA was synthesized using random priming, followed by end repair and 5′ phosphorylation. DA-tailing was added and adaptors with indexes were ligated. Ligation product was amplified using PCR, products between 300 and 500 bp were purified using AMPure beads (NEB) and amplified by PCR. Libraries were quantified with PicoGreen and QC with Bioanalyzer before analyzed by Illumina Hiseq2000 platform.

Amplicon libraries were prepared using the Ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies, Oxford, UK) and PCR barcoding kit (EXP-PCR096; Oxford Nanopore Technologies, Oxford, UK). Briefly, the protocol included end preparation, barcoding and sequencing adapter ligation. A total of 200 ng of purified extracted eDNA of each sample was end-repaired using NEBnext Ultra II End Repair Kit (New England Biolabs, Ipswich, MA, USA). This was cleaned using 1X AmPure beads (Beckman Coulter, Brea, CA, USA). Following this, barcode adapter ligation was performed with NEB blunt/TA ligase (New England Biolabs, Ipswich, MA, USA) and again cleaned with 1X AmPure beads. The barcode adapter-ligated products were quantified using Qubit Fluorometer (Thermo Fisher Scientific, USA). Barcoded samples were cleaned up using 1.6X AmPure beads and pooled at equimolar concentrations. Pooled barcoded samples were end prepared using NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). End-repaired DNA was cleaned up with 1X AmPure beads. The products were then adapter-ligated (AMX) using NEB blunt/TA ligase (New England Biolabs, Ipswich, MA, USA). The library mix was finally cleaned using AmPure beads and eluted in 15 µL elution buffer.