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.A: 实验描述：本RNA测序旨在对比细菌单培养与共培养组的基因表达差异。取发酵批次S和SL中18小时的发酵乳等分试样（4 mL）进行三次梯度稀释，随后在500 g、4℃条件下离心20分钟以去除凝固酪蛋白（重复该操作三次）；将上清液以5000 g、4℃离心30分钟两次，收集细菌细胞沉淀。完成总RNA的分离与纯化后，对每份样品中约5 μg总RNA进行核糖体RNA（ribosomal RNA, rRNA）去除，将残留RNA片段化并进行加A尾处理，连接带有T碱基突出端的cDNA连接接头，随后进行片段大小筛选并通过PCR扩增。最终构建的cDNA文库平均插入片段长度约为300 bp。最后，使用Illumina HiSeq 4000测序系统（LC Bio，中国杭州）开展150 bp双端测序。 B: 实验描述：本RNA测序旨在对比添加半胱氨酸组与未添加半胱氨酸组的基因表达差异，以解析半胱氨酸对细菌生长及γ-氨基丁酸（gamma-aminobutyric acid, GABA）生成的调控作用。将短乳杆菌（Lb. brevis）145接种于含5 g/L葡萄糖、2 g/L MSG的0.9%生理盐水中，分别添加与不添加半胱氨酸，培养24小时。按照制造商说明书，使用Ambion RiboPureTM细菌RNA提取试剂盒从两个独立批次的样品中提取总RNA。将约3×10^8个细菌细胞重悬于350 μL RNAwiz裂解液中，剧烈涡旋10~15秒；加入250 μL预冷氧化锆微珠，通过涡旋适配器涡旋10分钟完成细胞裂解。将裂解液与0.2倍体积的氯仿混合，离心收集含RNA的水相。将水相裂解液与0.5倍体积的100%乙醇充分混匀，随后过过滤柱：RNA被吸附于过滤柱基质上，杂质通过洗脱液去除。使用预热洗脱液洗脱得到总RNA，随后按照供应商说明书进行脱氧核糖核酸酶I（DNase I）处理，以去除污染的基因组DNA。使用QIAseq® FastSelect™ 多RNA去除试剂盒（细菌5S/16S/23S rRNA，货号335925）完成核糖体RNA去除。采用KAPA mRNA HyperPrep建库试剂盒构建cDNA文库：以500 ng总RNA为起始材料进行核糖体RNA去除，在NGS建库流程中通过QIAseq® FastSelect™试剂去除细菌5S、16S及23S rRNA。将处理后的RNA在镁离子存在下片段化至200~300 bp。以片段化且去除rRNA的RNA为模板，使用随机六聚体引物与反转录酶合成第一链cDNA。在第二链cDNA合成阶段，移除mRNA模板并合成替换链，形成平末端双链cDNA。对双链cDNA进行3’端腺苷酸化，并连接带索引的测序接头。通过12轮聚合酶链式反应（PCR）富集接头连接后的文库。将文库变性并稀释至最优浓度，使用Illumina NovaSeq 6000测序平台开展151 bp双端测序。