title: GSM1635877: DKS8.exo.1; Homo sapiens; miRNA-Seq
library_construction_protocol: Exosomes isolation: conditioned media centrifuged for 10 min at 300 X g, filtered through a 0.22um polyethersulfone filter, concentrated ~300-fold with a 100,000 MW cutoff centrifugal concentrator, high-speed centrifugation at 150,000 x g for 2 hours. RNA isolation:Cells grown serum-free for 24hrs (see growth protocol) or the isolated exosomes resuspended in PBS were subjected to RNA isolation through Trizol (Life Technologies)-chloroform extraction, followed by second RNA purification using RNA easy kit (Qiagen). Library prep was done  using NEB small RNA low input protocol using 6 ul sample input. Each sample was individually barcoded with NEB index primers for illumina. PCR amplification was done for 15 cycles and size selection was done using Ampure XP beads protocol as per manufactures instruction. .  Accurate quantification for sequencing applications was done using the qPCR-based KAPA Biosystems Library Quantification kit. Each library were diluted to a final concentration of 12.5nM and pooled equimolar prior to clustering.  Cluster generation will be carried out on a cBot v1.4.36.0 using Illumina's Truseq Single Read (SR) Cluster Kit v3.0. Sequencing was performed to generate approximately 15 million single end (SE) reads per sample on an Illumina HiSeq2500. Image analysis and base calling was performed using the standard Illumina Pipeline consisting of Real time Analysis (RTA) version v1.13 and demultiplexed using bcl2fastq converter with default settings.
attributes.tag: GEO Accession
attributes.value: GSM1635877