R1100 250

For the children with EA, fecal samples from three timepoints were evaluated: during PPI treatment, two weeks after (at one-year follow-up), and 1 month after discontinuation. All samples were sent by mail by the caregivers and stored at − 80° C within 2 days after collection.
In the ALADDIN study, feces were collected from the children at seven timepoints and frozen within 20 min of collection and stored at − 20 °C until later transported in a frozen state to storage at − 70 °C [64 (link), 65 (link)]. For the present study, only the 12-month sample was used.
All samples are preserved in DNA/RNA shield (Zymo Research—R1100-250), which keeps the bacterial constitution stable at room temperature for up to 30 days. All samples were shipped frozen to the Centre for Translational Microbiome Research on dry ice and stored at − 80 °C until processing.

Before the experiment, feces from 25-day-old control and treated piglets were collected from 10 piglets per group. The same procedure, using 10 piglets per group, was used at the end of the experiment (day 61 of the piglets' life) to generate representative samples of feces content from both animal groups. The DNA from each sample was kept in DNA/RNA Shield 1:10 (R1100-250, Zymo Research, USA) at −70°C before DNA extraction. DNA was extracted with a fecal DNA MiniPrep kit (D6010, Zymo Research, USA). Library preparation, metagenomic sequencing, and taxonomic characterization of reads was performed as described previously (21 (link)). ZymoBIOMICS Microbial Community Standard (D6300, Zymo Research, USA) was used as a microbiome profiling quality control. The results of taxonomic classification were visualized using the interactive online platform (https://genome-explorer.com).

Before the experiment, faeces from 25-day old piglets from each group (10 piglets per group) were collected. The same procedure, using 10 piglets per group, was performed at the end of the experiment (day 61 of the piglets’ life). Faecal samples from each of the group were pooled making 4 samples before and 4 samples after the experiment. Therefore, 8 samples representing 4 tested groups (before and after the experiment) were sequenced for microbial profiling. Specimens were kept in DNA/RNA Shield diluted 1:10 (R1100-250, Zymo Research, Irvine, CA USA) at −80 °C before DNA extraction. DNA was extracted with a faecal DNA MiniPrep kit (D6010, Zymo Research, Irvine, CA USA). Library preparation, metagenomic sequencing and taxonomic characterisation of reads was performed as previously described [28 (link)]. ZymoBIOMICS Microbial Community Standard (D6300, Zymo Research, Irvine, CA USA) was used as a microbiome profiling quality control. The results of taxonomic classification were visualised using the interactive online platform https://genome-explorer.com.