title: 16S rRNA V3-4 of biofilm at 5 degC with cold shock
design: Total DNA from the samples was extracted using the CTAB method, and its concentration was monitored on 1% agarose gel. The DNA concentration was adjusted to 1 ng/L with sterile water. a. PCR reactions were carried out using the Phusion High-Fidelity PCR Master Mix (New England Biolabs) protocol and materials, which used 15 L of the solution per sample, 2 M of forward and reverse primers, and 10 ng of the template DNA. Thermocycling consisted of initial denaturation (1 min at 98 C), followed by 30 cycles of denaturation (10s per cycle at 98 C), annealing (30 min at 50C), extension (30s at 72C), and final extension (5 min at 72C). The PCR products were then purified with a QIAquick Gel Extraction Kit (Qiagen, Germany). Library generation and sequencing were performed at Novagene (Beijing, China). The sequencing library was generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) and quality checked with a Qubit 2 Fluorometer (Thermo Fisher Scientific, USA) and Agilent Bioanalyzer 2100 system. The library was sequenced on the Illumina NovaSeq 6000 platform and generated a fragment library of 250 bp paired-end reads.