title: GSM1936972: Small 4 (small RNA seq); Bos taurus; ncRNA-Seq
library_construction_protocol: total RNA isolated via trizol Extracellular vesicle RNA (ranging from 1.8 ng – 100 ng) was used to initiate the TruSeq Small RNA library preparation protocol (Illumina #RS200-0012 kit A).  The EV RNA was ligated with 3’ and 5’ RNA adapters followed by a modified reverse transcription reaction and modified PCR amplification. Due to low starting RNA quantities, the reverse transcription of the RNA adapter ligated samples was modified by performing two duplicate reactions containing 6 μl of the 3’/5’ RNA ligated RNA. The 12.5 μl yield of each duplicate reverse transcription reaction was then pooled to obtain 25 μl of homogeneous cDNA.  The subsequent PCR amplification, with index adapter incorporation, was modified by replacing 8.5 μl of ultra-pure water in the PCR master mix with 8.5 μl of the reverse transcribed and pooled cDNA (21 μl cDNA total).  The modified PCR reaction was performed with 15 cycles of amplification.  Size selection and purification of the cDNA library construct was conducted using 3 % marker H gel cassettes on the Pippin Prep size fractionation system (Sage Science).  The Agilent 2100 Bioanalyzer was used with the High Sensitivity DNA kit (Agilent #5067-4626) or the DNA1000 kit (Agilent #5067-1504) to validate the purified libraries.  Libraries were quantified on the Illumina ECO Real Time PCR System using KAPA SYBR Universal Library Quant kit – Illumina (KAPA Biosystems KK4824).  Following quantification, libraries were adjusted to a 2 nM concentration and pooled for multiplexed sequencing.  Libraries are denatured and diluted to the appropriate pM concentration (based on qPCR results) followed by clonal clustering onto the sequencing flow cell using the TruSeq Rapid Single Read (SR) Cluster Kit-HS (Illumina GD402-4001).  The clonal clustering procedure was performed using the automated Illumina cBOT Cluster Station.  The clustered flow cell was sequenced on the Illumina HiSeq 2500 Sequencing System in Rapid Read mode with a 1x50 cycle read and index read using the TruSeq Rapid SBS kit-HS (Illumina FC402-4002).  Following collection, sequence data was converted from .bcl file format to FASTQ files and de-multiplexed into individual sequences for further downstream analysis.
attributes.tag: GEO Accession
attributes.value: GSM1936972