title: GSM2579631: Unaffected coral tissue from Waiopae, Hawaiʻi (Mcap_WU6); coral metagenome; RNA-Seq
library_construction_protocol: Total RNA was extracted from ~0.1g tissue powder using a combination of TRIzol reagent (Life Technologies) and the RNeasy Mini Kit (Qiagen). TRIzol was added to sample tubes (1ml per 0.1g tissue) and tubes were incubated for 5min at room temperature. Samples were then centrifuged at 12,000g at 4˚C for 10min, and the supernatant transferred to an RNase-free 1.5ml tube. Chloroform (Sigma-Aldrich) was added (0.2ml per 1ml TRIzol), the samples were shaken vigorously for 20–30 seconds, and then incubated at room temperature for 3 minutes. Samples were then centrifuged at 18,000g at 4˚C for 18 minutes. The top layer of aqueous solution was transferred to an RNase-free tube and an equal volume of 200 proof molecular grade ethanol (Sigma-Aldrich) was added. RNA was purified using the RNeasy Mini Kit with a 25min DNA digestion step (Qiagen RNase-free DNase Set) and eluted in 60µl DEPC-treated water (Omega). RNA quality and quantity was determined using the Qubit RNA Broad Range Assay Kit (Life Technologies) and Agilent RNA 6000 Nano Kit (Agilent), and RNA aliquots were stored at –80˚C. mRNA is purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C.  Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries.  The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX. Samples with a yield of ≥0.5 ng/ul are used for sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2579631