SUPPORTING DATA FOR "IMPROVING GAMMA-AMINOBUTYRIC ACID PRODUCTION IN MILK BY LEVILACTOBACILLUS BREVIS"
A: Description: The RNA sequencing was conducted to compare the gene expression differences between bacteria monoculture and co-culture groups. Aliquots (4 ml) of fermented milk in the S and SL fermentation batches at 18 h were diluted three times, followed by centrifugation at 500 g and 4°C for 20 min to remove coagulated caseins (three times), and supernatants were centrifuged twice at 5,000 g and 4°C for 30 min to collect bacterial cell pellets. After isolation and purification of total RNA, rRNA from approximately 5 ug of total RNA in each sample was depleted, and the residual RNA was fragmented to create A-tailing, cDNA-ligating adapters with a T-base overhang, followed by size selection of fragments and PCR amplification. The average insert size for the final cDNA library was about 300 bp. Finally, 150-bp paired-end sequencing was performed on an Illumina HiSeq 4000 system (LC Bio, Hangzhou, China).
B: Description: The RNA sequencing was conducted to compare the gene expression differences between cysteine group and without cysteine group to analyze the effect of cysteine on bacteria growth and GABA production. Lb. brevis 145 was inoculated in 0.9% saline containing 5 g/L glucose and 2 g/L MSG with or without cysteine for 24 h. Total RNA was extracted from the two different batches using Ambion RiboPureTM-Bacteria kit following the manufacturer’s instructions. The bacterial cells (approximately 3×108) were resuspended in 350 μL RNAwiz by vortexing vigorously for 10-15 sec, lysed using 250 μL of ice cold Zirconia beads for 10 min on a vortex adapter. The lysate was mixed with 0.2 volumes of chloroforms and centrifuged to collect the RNA containing aqueous phase. The aqueous lysate was mixed well with 0.5 volumes of 100% ethanol, which was then passed through filter cartridge. RNA was trapped in the filter and impurities were removed by wash solutions. Next, RNA was eluted by preheated elution solution. DNase I treatment was also performed to remove the contaminating DNA as outlined by the supplier.Ribosomal RNA (rRNA) depletion was performed using QIAseq® FastSelect™ Multi-RNA Removal Kit (Bacterial 5S/16S/23S rRNA, Cat# 335925). cDNA libraries were prepared by by KAPA mRNA HyperPrep Kit. Five hundred nanogram of total RNA was used as starting material for ribosomal RNA depletion. Bacterial 5S, 16S and 23S rRNA were removed by the QIAseq® FastSelect™ reagents during the NGS library preparation. The processed RNA was fragmented to 200-300 bp in the presence of magnesium ions. The fragmented rRNA-depleted RNA was then applied as template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed, and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent 3’adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 12 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing.