Computational protocol: Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon

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Protocol publication

[…] Paired biopsy samples from the proximal and distal colon were collected from a total of 19 healthy controls (9 women, 10 men; mean age: 59.7 ± 10.2 years) with no known autoimmune disease and/or malignancies. The biopsy tissues were immediately embedded in RNAlater (Ambion) and snap-frozen at −80°C. DNA was extracted using the FastDNA SPIN Kit for Soil in conjunction with a Fastprep-24 machine (MP Biomedicals, Santa Ana, CA) following the manufacturer’s protocols.The V1–V2 regions of bacterial 16S rRNA genes were PCR amplified using Q5 Taq polymerase (New England BioLabs, Ipswich, MA) and Golay barcoded primers (purchased from Eurofins MWG Operon, Huntsville, AL) MiSeq-27F (5′-AATGATACGGCGACCACCGAGATCTACACTATGGTAATT CCAGMGTTYGATYMTGGCTCAG-3′) and MiSeq-338R (5′-CAAGCAGAAGACGGCATACGAGAT-barcode-AGTCAGTCAGAAGCTGCCTCCCGTAGGAGT-3′). Each sample included in the study was amplified with a 338R primer containing a unique 12-mer Golay barcode sequence. The PCR conditions were as follows: 98°C for 2 minutes followed by 25 cycles of 98°C for 30 seconds, 50°C for 30 seconds, 72°C for 1 minute and 30 seconds, and then a final extension step at 72°C for 5 minutes. Four PCR reactions per sample were performed, which were then pooled into a single amplicon mix for each of the samples. The PCR amplicons from each sample were then quantified using a Qubit 2.0 Fluorometer (Invitrogen/Life Technologies), and equimolar concentrations of each sample were added together into a final mastermix for sequencing on an Illumina MiSeq machine (2 × 250 bp read length) (Illumina, San Diego, CA).Sequence data have been deposited in the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under study number ERP007146. The individual accession numbers for each of the biopsy samples are shown in .The resulting sequence data was processed using the mothur software package (http://www.mothur.org/), closely following their MiSeq SOP. In brief, paired contigs were created from the forward and reverse MiSeq reads, and all contigs shorter than 260 bp (base pairs) and longer than 450 bp, and those containing any ambiguous bases or homopolymeric stretches of longer than seven bases were removed. Chimeras were removed using Perseus as implemented in mothur. Operational taxonomic units (OTUs) were generated from preclustered data (diffs = 3) at the 97% cutoff level and then assigned a taxonomic classification using the Ribosomal Database Project (RDP) taxonomy provided with mothur. Diversity measures such as Shannon and inverse Simpson indices were calculated in mothur, where every sample was first subsampled down to 450 reads to ensure equal sequencing depth for these comparisons. Good’s coverage (an estimate of completeness of species sampling) at this sequencing depth was on average 93.4%. A cluster dendrogram using the Bray Curtis calculator was generated in mothur and visualized using the iTOL online resource (http://itol.embl.de/). Differences in bacterial community structure between proximal and distal colonic biopsies were assessed using the analysis of molecular variance (AMOVA), parsimony, Metastats, and LEfSe tests as implemented in the mothur software package. […]

Pipeline specifications

Software tools mothur, Perseus, iTOL, Metastats, LEfSe
Databases ENA
Applications Phylogenetics, Metagenomic sequencing analysis
Organisms Homo sapiens, Mus musculus