Transcriptome sequencing of murine myeloid leukemia genome
Experimental procedures. mRNA Sequencing of polyadenylated RNA (mRNA-seq) was performed using total RNA isolated from flash frozen spleen specimens using mirVana™ miRNA Isolation Kit. 5 μg of total RNA isolated from D5 leukemic spleen was mixed with ERCC RNA Spike-In was used for poly-A enrichment using Dynabeads® mRNA DIRECTTM Micro Purification Kit. 200 ng of poly-A-enriched mRNA was used for library construction according to the Ion Total RNA-Seq Kit v2, protocol for non-barcoded libraries, Ion Sphere™ Particle (ISP) loading using Ion OneTouch™ 2 System and sequencing on the Ion Proton™ platform with PIv2 semiconductor wafer chips per manufacturers recommended instructions. Each RNA-seq library was sequenced individually on a PIv2 chip to achieve >4 GB (~70 million reads) per specimen. After quality control checks, raw sequencing read files were generated by Ion Torrent Suite.
Houston Methodist Research Institute
D0005_SP_mRNA-Seq_T