title: GSM2308777: Red tide rep3; Trichechus manatus latirostris; RNA-Seq
library_construction_protocol: Sample preparation included RNA extraction from buffy coats, purification using DNase and column treatments to remove residual DNA contamination, depletion of rRNA and globin using EpiCentre ScriptSeq™ kits, and creation of cDNA libraries following standard protocols (EpiCentre, Madison, WI, USA). RNAlater (Life Technologies™, Carlsbad, CA, USA) was used to preserve the buffy coat samples in a ratio of 1.2 mL RNAlater to <0.5 mL buffy coat according to the manufacturer’s protocol (Life Technologies™). The samples were stored at -20 °C in RNAlater for approximately 9 months. RNA was extracted from manatee buffy coat samples according to the Tel-Test Inc. method for total RNA extraction using Stat-60 in a 1:10 ratio of buffy coat sample to Stat-60 (Tel-Test, Inc., Friendswood, TX) For RNA processing and sequencing library preparation, the Illumina® EpiCentre ScriptSeq™ Complete Gold (Blood)-Low Input Kit (Illumina® EpiCentre, Madison, WI, USA) was used along with the Illumina® FailSafe™ PCR Enzyme Mix and ScriptSeq™ Index PCR primers (set 1). The kit protocols were followed with minor modifications. This kit’s ‘Globin and Ribo-Zero’ reagents were designed to remove globin and rRNA from RNA samples (EpiCentre ScriptSeq™, Madison, WI). The Zymo Research (Irvine, CA, USA) RNA Clean and Concentrator-5 was used to purify RNAs (>200 nt) and concentrate the depleted samples. Individual libraries were made for each of the samples.
attributes.tag: GEO Accession
attributes.value: GSM2308777