Qubit dsdna high sensitivity assay

One mL aliquots of HM were centrifuged at 40,000× g for 5 min at 4 °C. The supernatant and lipid fraction were discarded. Maternal faecal, infant faecal, and infant oral samples were centrifuged at 40,000× g for 5 min at 4 °C and the supernatant was discarded. For all sample types, DNA was extracted from the cell pellet using the QIAGEN MagAttract Microbial DNA Isolation Kit (QIAGEN, Hilden, Germany) on the Kingfisher Flex platform, following the manufacturer’s instructions. Two negative extraction controls each consisting of 1 mL sterile nuclease-free water (Integrated DNA Technologies, Coralville, IA, USA) were included at the centre of each 96-well extraction plate.
Total DNA yield was assessed using the Qubit^® dsDNA High Sensitivity Assay (Invitrogen, Mulgrave, VIC, Australia) on a Qubit^® 2.0 Fluorometer (Life Technologies, Mulgrave, VIC, Australia) according to the manufacturer’s instructions. The limit of detection was 10 pg/μL.

Blood samples on FTA cards (Whatman Inc., Clifton, NJ, United States) were collected from 65 Brazilian families (with confirmed kinship) composed by mother, father, and two children (260 individuals in total), as well as from 84 unrelated Brazilian individuals. Informed consent was obtained from all participants included in the study. The project was approved by an ethical committee of the State University of Rio de Janeiro (CAAE: 0067.0.228.000-09).
DNA was extracted with the standard phenol-chloroform method. DNA extract concentration was measured using the InnoQuant HY kit (InnoGenomics) according to the manufacturer’s protocol or using the Qubit dsDNA High Sensitivity assay and the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions.

For Sanger sequencing, target sites were PCR amplified (Table S1) using PrimeStar GXL polymerase (Takara), and PCR cleanup was done using ExoSap-IT Express (Thermo Fisher). Chromatograms were analyzed using EditR to determine base editing efficiencies.⁴⁹ (link) Candidate OTSs were identified with CRISPOR, and the top five sites, by cutting frequency determination (CFD) score, for which PCR products were successfully obtained were selected.⁵⁰ (link)^,51 (link) Target sites were PCR amplified (Table S1) using PrimeStar GXL polymerase (Takara), and a second round of PCR was used to add Illumina flow cell binding sequences and barcodes. PCR products were purified with AMPure XP beads (Beckman Coulter), analyzed for integrity on a 2200 TapeStation system (Agilent), and quantified by Qubit dsDNA high-sensitivity assay (Invitrogen) before pooling and loading onto an Illumina MiSeq. Following demultiplexing, resulting reads were analyzed with CRISPResso2 for editing frequency.⁵² (link) To analyze the number of AAV integration events at the on-target site, we followed a previously established method.⁵³ (link) Sequencing files were aligned to AAV vector sequences using the bwa program (version 0.7.17) and sorted with samtools (version 1.6), and the number of reads that had vector sequences were counted and normalized to number of reads that mapped to the target amplicon.

Bacterial DNA was extracted from 1 mL human milk samples using the QIAGEN MagAttract Microbial DNA Isolation Kit (Qiagen, Chadstone, Australia) on the Kingfisher Flex platform following the manufacturer’s instructions, as described previously [107 (link)]. Total DNA yield was assessed using the Qubit^® dsDNA High Sensitivity Assay (Invitrogen, Mulgrave, VIC, Australia) on a Qubit^® 2.0 Fluorometer (Life Technologies, Mulgrave, VIC, Australia) according to manufacturer’s instructions. The limit of detection was 10 pg/μL.

Combinations of different commercially available DNA extraction kits were used, including QIAamp DNA Micro Kit (Qiagen 56304), GenSolve (Gen Vault, GVR110), NucleoSpin (gDNA clean-up, Macherey-Nagel 740230), and the extra-small (XS) version of NucleoSpin (gDNA clean-up XS, Macherey-Nagel 740904). Reported quantifications were done using Nanodrop, unless indicated otherwise using Qubit™ dsDNA High Sensitivity Assay, Invitrogen Q32851. Detailed protocols are included in Additional Methods (Additional File 2).

For experiments in which purified genomic DNA was subjected to DNA-IP, bulk DNA quantification was performed using the Qubit dsDNA high-sensitivity assay (Invitrogen, Thermo Fisher Scientific). The Qubit assay was conducted using a Life Technologies (Thermo Fisher Scientific) Qubit 3.0 Fluorimeter according to the manufacturer’s instructions.