GSM4508925
GSM4508925: ALDHnegative_PD901_rep1; Homo sapiens; OTHER
Cells were labeled with 2 mM Bromouridine (Sigma Aldrich) for 30 min, sorted, total RNA isolated by using TRIzol reagent (Invitrogen), and Bru-labeled RNA was isolated from the total RNA by incubation with anti-BrdU antibodies (BD Biosciences) conjugated to magnetic beads (Dynabeads, Goat anti-Mouse IgG; Invitrogen) under gentle agitation at room temperature for 1 h. For more detail, see Paulsen et. al 2013 (Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response. Proc. Natl. Acad. Sci. U.S.A. 2013; 110:2240–2245) To equal amounts of total RNA, a spike cocktail was added, consisting of a mixture of Bru-labeled and unlabeled E. coli, drosophila and synthetic RNAs. To isolate the Bromouridine incorporated RNA (Bru-RNA), total RNA was incubated at 80˚C for 10 minutes, then added to magnetic goat anti-mouse IgG Dynabeads (Invitrogen) conjugated with anti-BrdU antibodies (Becton Dickinson). The RNA and beads were incubated for 1 hour at room temperature with gentle mixing, followed by 3 washes with 0.1% BSA in PBS. The beads were resuspended in 35μl DEPC-treated water, mixed and incubated at 96˚C for 10 min to elute the Bru-RNA. The eluted Bru-RNA was quantitated and used in the preparation of cDNA libraries. Briefly, 300ng of Bru-RNA was fragmented, and rRNA eliminated by mixing with first strand buffer,random primers and FastSelect (Qiagen)and incubated at 85˚C for 10 min. The first strand cDNA was synthesized in the presence of Actinomycin D to result in strand specific libraries and was purified using AMPure RNAclean beads (Beckman Coulter). The second strand cDNA was synthesized, then purified using AMPure XP beads (Beckman Coulter). Subsequently, end repair, adenylation, and adaptor ligation was performed, followed by a size selection. For this, the cDNA was run on a 3% NuSieve 3:1 agarose gel (Lonza) and gel slices excised in the 500bp region. The gel slices were purified using the QIAEX II Gel Extraction Kit (Qiagen) and then PCR amplified using dual index primers. Resulting libraries were purified with AmPure XP beads, quantitated and submitted to the University of Michigan Advanced Genomics core for sequencing.
GEO
GSM4508925
GEO Accession
GSM4508925