Experiment : 2
The point of this experiment was to examine whether or not tonic 5nM dopamine could produce global changes in microRNA (miR) expression in the spiny lobster, Panulirus interruptus. Six animals were used in this experiment. The bilateral ring of thoracic ganglia was dissected from each animal and cut in half. One half was superfused with saline and the other half with saline containing 5nM dopamine. After 1hr, the tissue was flash frozen in liquid nitrogen. RNA was isolated from the saline and dopamine treatment groups with a mirVana kit, and the twelve samples (6 saline & 6 dopamine) were sent to LC Sciences. LC Sciences generated a small RNA cDNA library for each sample and performed deep sequencing using an Illumina platform. Unmappable sequences were removed from raw reads, and sequences ranging from 15-32 bases in length were analyzed further. Identical sequences were clustered and unique reads were mapped to various databases. Reads that mapped to mRNA, RFam and RepBase were not analyzed further. Reads that mapped to miRBase and hairpin structures in arthropod genomes were assumed to be true miRs and were included here. Other small RNAs that did not map to miRBase and mapped to genomic sequences that did not have the propensity to form hairpins, or were not found in any database, were not included here. Unique sequences representing putative lobster miRs were designated pint-mirs. The pint-mirs were further grouped into families based on known homology. For example, 3 unique sequences mapping to ame-mir-2-3, nvi-mir-2b and dpu-mir-2-1 in miRBase were all grouped into the miR-2 family. Unique members of the same family were indicated by a lower case letter(s), e.g., pint-mir-2-a, pint-mir-2-b, etc. Lower case letters do not imply orthology. The 88 putative pint-miRs with no known homology were named according to their read IDs. In the majority of cases, pint-mirs have not been experimentally validated and so are referred to as putative mirs.
The following numbers represent the summed totals for all 12 small RNA libraries:
- raw reads: : 100,980,705
- mappable reads: 83,280,252
- reads mapped to mRNA, RFam, RepBase: : 40,693, 592
- reads mapped to miRBase: 24,802,440 ***
- reads not mapped to miRBase but mapped to genomic sequences with the propensity to form hairpins: 8,904 ***
- reads not mapped to miRBase, but mapped to arthropod genomic sequences with no propensity to form hairpins: 204,039
- reads that represent small RNAs in P. interruptus, but cannot be further classified because they were not found in any database: 19,571,277
The putative miRs for this experiment can be visualized here. The number of reads for each pint-mir in a given animal is listed. The animals were numbered 1-6. The letter S or D after the number indicates saline or dopamine treatment. This experiment has been deposited in xx and the accession number is xx. It is associated with the following publications...
