title: Illumina MiSeq sequencing; Opioid exacerbates gut dysbiosis in a murine model Citrobacter rodentium infection
design: Opioid exacerbates gut dysbiosis in a murine model Citrobacter rodentium infection
library_construction_protocol: Stool samples were collected into 1.7ml RNase/DNase-free tubes (Catalog #: C-2170, Denville Scientific, Holliston, MA, USA) at time points accordingly. The fecal samples were immediately frozen on dry ice and then stored at -80 C C. rodentium strain (DBS 100) was obtained from American Type Culture Collection (ATCC; Manassas, VA). Nalidixic acid resistant C. rodentium was selectively screened out on LB agar plate with 20ug/ml Nalidixic acid. A spontaneous mutant of C. rodentium DBS100 resistant to nalidixic acid (20μg/ml) would be obtained by growing DBS100 at 37°C in LB medium supplemented with nalidixic acid at 5μg/ml for 7 h before spreading the liquid culture onto LB agar plates supplemented with nalidixic acid at 20μg/ml. The selective LB agar plates and broth would be made by adding nalidixic acid up to 20μg/ml in order to selectively culture C. rodentium in the future study 21. The nalidixic acid solution was made with Mili-Q water in fume hood and the stock solution was sterilized through a 0.22um filter using a 10-ml syringe with in the sterile hood. Bacterial culture on blood agar plates without antibiotics or LB agar plates with 20 µg/ml Nalidixic acid over night at 37 °C.  Mice received morphine through the pellet implantation method as described (Bryant et al. 1988). Using this method, plasma levels of morphine were maintained in the 0.6-2.0-microg/ml range (range observed in opioid abusers and patients on opioids for moderate to severe pain). Furthermore, this model is commonly used in the study of opiate dependence and addiction(Bryant et al. 1988). Briefly, placebo or 25 mg morphine pellets (National Institutes of Health [NIH]/National Institute on Drug Abuse [NIDA], Bethesda, MD) were inserted in a small pocket created by a small skin incision on the dorsal side of animals; incisions were closed using surgical wound clips (Stoelting, 9 mm Stainless Steel, Wooddale, IL). Mice were subcutaneously treated with placebo or 25 mg morphine pellet for 24 hours prior to infection. Mice were infected with 1x10^9 C. rodentium in 200 ul medium via oral gavage. Mice were sacrificed for study at day 5 post infection with C. rodentium. DNA extractions from fecal matters were carried out using the PowerSoil DNA isolation kit (Catalog #: 12888-100, MO BIO Laboratories, Carlsbad, CA, USA). All extracted DNA samples were stored at -80C until amplification. Fecal DNA samples (25 ng) were used as template for PCR amplification of the V4 region of the 16S rRNA gene. Degenerate primer sets were designed with Illumina index sequences on the 5 end of the reverse primer, which were specific to each fecal DNA sample and allowed for multiplexed sequencing. Primers also contained Illumina PCR primer sequences (reverse primer) and Illumina TruSeq Universal Adapter sequences (forward primers) for library creation. 1) Primer Sequences (16S-specific portion in bold) Meta_V4_515F: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA Meta_V4_806R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT The indexing primers are as follows. This step adds both the index and the flowcell adapters. [i5] and [i7] refer to the index sequence codes used by Illumina. The p5 and p7 flow cell adapters are in bold. Forward indexing primer: AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC Reverse indexing primer: CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG 2) Cycling Conditions -PCR reactions are performed using KAPA HiFidelity Hot Start Polymerase PCR 1 (using the Meta_V4_515F/Meta_V4_806R primer pair) 95C 5 minutes 20 cycles of: 98C 20 seconds 55C 15 seconds 72C 1 minute Hold at 4C -After the first round of amplification, PCR 1 products are diluted 1:100 and 5 ul of 1:100 PCR 1 is used in the second PCR reaction. PCR 2 (using different combinations of forward and reverse indexing primers) 95C 5 minutes 10 cycles of: 98C 20 seconds 55C 15 seconds 72C 1 minute Hold at 4C
attributes.tag: Experimental Factor: compound
attributes.value: placebo
attributes.tag: Experimental Factor: infect
attributes.value: none