Diethylpyrocarbonate treated water

The total RNA was sent to Beijing Genomics Institute (BGI) (Shenzhen, Guangdong province, China), small RNAs were sequenced using Illumina HiSeq™ 2000 high throughput sequencing technology. sRNAs with the length of 18-30 nt were first separated from the total RNA by running on 15% (w/v) TBE urea polyacrylamide gel. The excised gel with sRNAs from 18 to 30 nt in length were submerged in 600 μL of 0.3 M sodium chloride overnight at 4°C. The sRNA fragments were dissolved in diethylpyrocarbonate treated water (Ambion, Austin, TX, USA). The sRNAs were then added 5′ adaptor by T4 RNA ligase (Takara, Dalian, China) in the presence of RNase OUT Ribonuclease Inhibitor (Invitrogen, China) overnight at 4°C according to the manual. After obtaining the 5′ ligation products, a 3′ adaptor was added to the selecting 5′ adaptor sRNAs following the same procedures as the 5′ adaptor ligation. Finally, both 3′ and 5′ adaptors ligated sRNAs were run on a 10% (w/v) TBE urea polyacrylamide gel, then the 44 nt RNAs were excised. The sRNA was reversely transcribed to cDNA with the RT primer by Superscript II reverse transcriptase (Invitrogen, China). The cDNA was further quantified by Agilent 2100 and sequenced by the Illumina 1G Genome Analyzer.

Total RNA was extracted from ARPE-19 cells according to the manufacturer’s protocol (TRIzol Reagent; Invitrogen-Gibco). For cDNA synthesis, total RNA (2 μg) was reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Subsequently, quantitative polymerase chain reaction (qPCR) for p62 was performed according to the protocol of the TaqMan assay. Briefly, the qPCR was performed in triplicate, using 8-tube strips with 1 μL of TaqMan probe, 10 μL of master mix (TaqMan Universal Master Mix II, Applied Biosystems), 4 μL of cDNA (100 ng), and 5 μL of diethylpyrocarbonate-treated water (Invitrogen, Carlsbad, CA, USA). The thermal cycling profile consisted of an initial temperature of 50 °C for 2 min, followed by a denaturation step at 95 °C for 10 min, and then 40 successive cycles at 95 °C for 15 s and 60 °C for 1 min. The primer set of the TaqMan assay for SQSTM1/p62 was Hs01061917_g1 (Applied Biosystems). The amount of target mRNA relative to the expression of the endogenous control gene and relative to values from the control group was calculated using the relative quantitation method. mRNA expression levels were normalized using the expression of GAPDH (Hs02758991_g1, Applied Biosystems) as the endogenous housekeeping gene.

RNA was extracted from serum samples using TRI Reagent (SIGMA-ALDRICH, USA), according to the manufacturer's instructions. The RNA was eluted in diethylpyrocarbonate-treated water (Invitrogen, Carlsbad, CA, USA).
One-step standard RT-PCR was performed using a Transcriptor One-Step RT-PCR Kit (Roche Diagnostics GmbH) according to the manufacturer's instructions using the primers pair (324F-5'-ATG CCC WTA GTA GGA CTA GCA-3'; 326R-5'-TCA ACT CCA TGT GCC ATG TAC-3'), designed to amplify a 288-bp length fragment from the 5′UTR region of Pestivirus genome (32 (link)). Additional primers pair (B32-5′-TGC TAC TAA AAA TCT CTG CTGT-3′; B31-5′-CCA TCT ATR CAY ACA TAR ATG TGGT-3′) was designed to amplify a 441-bp length fragment from the N^pro region of BVDV (33 (link)). The final volume of RT-PCR reaction mixture was 25 μl including 23 μl of reaction mix and 2 μl of RNA template. The amplification of 5′UTR region was done at 50°C for 30 min and 94°C for 7 min, followed by 10 cycles of 94°C for 10 s, 53°C for 30 s, 68°C for 30 s, then 25 cycles of 94°C for 10 s, 53°C for 30 s, 68°C for 33 s with a final extension step at 68°C for 7 min, while the thermal profile for N^pro region was similar except for the annealing temperature decreased to 50°C.
The RT-PCR products were submitted to electrophoresis in 1.5% agarose gel in TBE buffer, stained with ethidium bromide, and visualized under UV light.

Biofilm DNA was extracted using the DNeasy PowerSoil DNA extraction kit (Qiagen, The Netherlands) by weighing ∼300 mg in a PowerBead tube. The manufacturer’s instructions were followed except for the homogenization step as this was done using a TissueLyser LT (Qiagen) at 50 Hz for 10 min. DNA was eluted in diethyl pyrocarbonate-treated water (Invitrogen, USA). Eluted DNA was stored at −20°C until sequencing. Due to difficulties during DNA extraction, biofilms from Leiden were not sequenced, and 16S rRNA gene amplicon sequencing was done by Macrogen (Macrogen, Inc., Korea) using the Illumina MiSeq, next-generation sequencing platform. Paired-end libraries were constructed using the Illumina Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 (Illumina, The Netherlands). Primers used for bacterial amplification were Bac341F (5′-CCTACGGGNGGCWGCAG-3′; Herlemann et al. 2011 (link)) and Bac806R (5′-GGACTACHVGGGTWTCTAAT-3′; Caporaso et al. 2012 (link)). Raw sequencing reads were deposited in the European Nucleotide Archive at the European Bioinformatics Institute under project number PRJEB62150 (https://www.ebi.ac.uk/ena/browser/view/ PRJEB62150).

Total RNA was extracted from tissues using TRIzol (Ambion, Austin, TX, USA). Pieces of distal colon tissues were treated with 800 μL of TRIzol reagent, homogenized using a pestle, and incubated at room temperature (RT) for 10 min. Then, 200 μL of chloroform was added and centrifuged at 12,000× g, 4 °C for 15 min. The upper layer was transferred to a microfuge tube, and an equal amount of isopropanol was added. After 10 min of incubation at RT, the mixture was centrifuged at 12,000× g, 4 °C for 10 min. Then, the supernatant was removed, and 1 mL of 75% ethanol was added. Finally, the mixture was centrifuged at 7500× g, 4 °C for 5 min, and the supernatant was removed. The RNA pellet was air-dried and dissolved in 20 μL of diethylpyrocarbonate-treated water (Invitrogen, Carlsbad, CA, USA). RNA quantity and quality were examined using an Infinite M200 PRO TECAN (Research Triangle Park, NC, USA). The isolated total RNA was stored at −80 °C until use.