Illumina MiSeq sequencing; Morphine treatment leads to temporal modulation of the gut microbiome
Morphine treatment leads to temporal modulation of the gut microbiome
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 Mice received morphine and pellet implantation method as described(Bryant et al. 1988). Using this method, plasma levels of morphine are in the 0.6–2.0-microg/ml range (range seen 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 or 30 mg naltrexone 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 animal's dorsal side; incisions were closed using surgical wound clips (Stoelting, 9 mm Stainless Steel, Wooddale, IL). 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 -80°C 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
Experimental Factor: morphine+naltrexone
compound
Experimental Factor: 1+10
dose
Experimental Factor: 0
time