Mid output kit

Metatranscriptome was studied for all the samples from the first experiment and for selected samples for the second experiment (Supplementary Table 3). Two replicates were sequenced for all samples (each replicate was obtained by pooling the RNA extracted from two different cheeses). Ribosomal RNA (rRNA) was depleted by using the Ribo-Zero Magnetic kit (Epicentre, Charlotte, North Carolina) and the mRNA enriched RNA was purified by Agencourt RNAClean XP (Beckman Coulter, Milano, Italy) following the manufacturer’s instruction. Then, library preparation and sample multiplexing were carried out by using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, Charlotte, North Carolina) (insert size around 300 bp). The quality of the library was checked by 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California). Sequencing was carried out on a Next Seq 500 Sequencer with the Mid Output Kit (both from Illumina, San Diego, California), yielding 150 bp single-end reads.

To characterize the bacterial community composition of the samples, DNA was extracted with the ZymoBIOMICS 96 DNA kit (product D4309) from Zymo Research using the manufacturer’s suggested protocol. Sequencing libraries were prepared using the Illumina Nextera kit and methods described in Baym et al. (64 (link)). Briefly, DNA was diluted to 0.5 ng/μl and added to 0.25 μl of Nextera enzyme and 1.25 μl of tagmentation buffer. This mixture was incubated at 55°C for 10 min and then placed on ice for the remainder of the protocol. Barcodes were added using Phusion polymerase (New England Biolabs), and excess adaptors were cleaned using AMPure XP (Beckman Coulter Life Sciences) magnetic beads. Quality and concentration were assessed using a PicoGreen assay (ThermoFisher), and the distribution of fragment sizes was determined using a Bioanalyzer. These libraries were loaded onto the Illumina Next-Seq 500 at 1.8 pM concentrations and sequenced using Illumina’s mid-output kit for 75-bp paired-end sequencing, resulting in a total of 144,023,583 reads and an average of 1,425,976 reads/sample (maximum, 5,902,966; minimum, 7) (Table S1).

Sequencing libraries were prepared using the Illumina Nextera kit and methods described in Baym et al. (64 (link)). Briefly, DNA from each sample was diluted to 0.5 ng/μl and tagmented with the Nextera enzyme (Illumina) for 10 min at 55°C. Following tagmentation, each sample received 1-μl forward and 1-μl reverse barcodes, which were added via PCR using Phusion DNA polymerase (New England BioLabs). After PCR, the libraries were cleaned of smaller DNA fragments, using AMPure XP magnetic beads (Beckman-Coulter), and pooled by concentration. Libraries were quantified using the Quanti-iT PicoGreen dsDNA kit (Thermo Fisher Scientific), and DNA was run on a gel to check fragment size. These libraries were loaded onto the Illumina Next-Seq 500 at 1.8-pM concentrations and Illumina’s midoutput kit for 75-bp paired-end sequencing.

SH‐SY5Y cells (2.2 × 10⁶) were cultured on 100 mm dishes for 24 h. The medium was then replaced with either 1 or 2 mM FeCl₂. Cells were passaged every 3–4 days by adding fresh FeCl₂‐treated media. After 2 weeks, 0.3 × 10⁶ cells were seeded in three wells of a 6‐well plate; the medium was replaced with fresh FeCl₂‐treated medium the next day. Non‐treated cells were prepared according to the same procedure of iron‐challenged cell culture but incubated in media without FeCl₂. When untreated or iron‐treated cells reached 70~80% confluency in the 6‐well plates, the medium was aspirated, and lysis buffer was added to each well. RNA from each well was isolated using a Purelink RNA mini kit (12183018A; ThermoFisher) according to the manufacturer's protocol. RNA integrity was verified via an RNA 6000 pico assay run on Bioanalyzer, and mRNA was isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module. From the mRNA, a transcriptome library was prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following the manufacturer's protocol. After evaluation of the length of the library, the iron‐treated and untreated libraries were sequenced using Illumina NextSeq 500/550 with 150 cycles mid‐output kit (Cat # 20024904; Illumina).