title: GSM5825383: MYCS62A_Splenomegaly_I170; Mus musculus; RNA-Seq
library_construction_protocol: Trizol extraction per manufacturer's protocol Sequencing libraries were constructed using a NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA) per manufactuer instructions. Briefly, poly(A)+ RNA was isolated from 500ng of total RNA (per sample) using oligo-dT-coated magnetic beads. The recovered RNA was then chemically fragmented. First strand cDNA was generated using random hexamers as primers for reverse transcriptase. Strand-specific second strand cDNA synthesis was performed and a single 'A' nucleotide was then added to each end. Illumina adaptors were ligated to the cDNAs. Limited-round (15-cycle) PCR was used to amplify the material to yield the final libraries.  Library concentration was determined using real-time PCR with primers complementary to the Illumina adaptors.  All libraries were prepared with indexing barcodes to permit multiplexing in a single lane. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols.