GSM3502793
GSM3502793: EGFR Cell_shortRNA; Mus musculus; ncRNA-Seq
Conditioned media was centrifuged at 300 x g for 10 minutes, then 2000 x g for 15 minutes. The cleared conditioned media was then passed sequentially through a series of syringe filters with decreasing pore size, 2 µm (Whatman), 0.8 µm (Millipore), 0.22 µm (Millipore), and 0.02 µm (Whatman) and the flow-through media cleared of EVs by filtration was further concentrated using a size-exclusion spin column centrifuged for 1 hour at 4200 x g using a 3 kDa size-exclusion spin column (Amicon, Millipore). This serial filtration series isolated small microvesicles (0.22 µm), exosomes (0.02 µm) and RNP complexes (3 kDa) respectively. Captured vesicles were lysed on the filter and total RNA was extracted using the miRCURY™ RNA Isolation Kit – Cell & Plant(Exiqon). RNA was rRNA-depleted using the Ribo-Zero rRNA Removal Kits (Illumina, CA) and one quarter was fragmented to 100–500 nt using the 5× First-Strand Buffer (Clontech, CA) and used in the SMARTer Stranded RNA-Seq Kit (Clontech) to generate the long RNA library.  The remaining rRNA-depleted samples were used in the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) to generate the small RNA library.  The libraries were sequenced on HiSeq 2000 (Illumina) with single read 50 cycles at the Beijing Genomics Institute (BGI, China).
GEO
GSM3502793
GEO Accession
GSM3502793