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All PCR#1 products for individual biological samples were pooled based on relative band intensity when 5 μl of PCR#1 reaction from a representative sample, non-diabetic 1, was analyzed on a 1.5% agarose gel.
According to their site of collection, the collected biological samples were pooled in three groups: (i) patients having pus and serositises, (ii) patient with urogenital infections (urine, vaginal, and urethral), and (iii) mucous membrane (skin and nostril) of healthy persons.
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Protein concentration was determined by Coomassie Plus reagent (Promega, Madison, WI) with a BSA standard curve read at 595 nm on an EL808 Microplate Absorbance Reader (BioTek, Winooski, VT. Two enrichments per biological sample were pooled to give 600 µg PSD protein.
Within each individual experiment, equal amounts of extracted RNA from biological replicate samples were pooled.
For each of the three biological replicates, these four samples were pooled and sequenced on their own Illumina lane with 50-nucleotide paired-end reass as described in Bloom (2014).
Due to sample scarcity, the biological duplicates for these membrane protein samples were pooled together to obtain enough protein for MS analysis.
Samples were pooled from three biological replicates each.
Before RNA and protein extraction, five samples were pooled for each biological replicate.
For all the tissues, three randomly chosen samples were pooled to form a biological replicate.
So in practice, different samples were pooled together to reduce the biological variation [ 29, 36- 38].
Individual libraries were prepared with barcode and all nine samples (biological triplicates of 0 hr, 2 hr, and 24 hr samples) were pooled for sequencing on one lane.
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