GSM2127900: DAP5 Tot b; Homo sapiens; RNA-Seq
100ug/ml cyclohexamide was add to the meduim for 10min. Plates were wash and harvest in LBA (20mM Tris ph7.5, 45mM KCl, 10mM MgCl2, 0.1mg/ml CHX, 1mM DTT, 2.5ul/ml RNasin, Protease inhibitor cocktail) with 1% NP-40. 12 O.D of the cleared lysate were loaded on 10-50% sucrose gradients in LBA and centrifuge for 100min at 39,000 rpm in SW-41 rotor (Beckman Optima LE-80k Ultracentrifuge). The gradients were collected with Teledyne ISCO (UA-6), 0.5ml fractions were collected. 30l of 10% SDS and 1ml of 100% Ethanol was added to each fraction. The fractions were incubated at -20ºC overnight, and centrifuged for 15min at 20,000g. The pellet was resuspended in Tri-reagent and RNA was purified according to the protocol. For total RNA, the same procedure was done directly on lysate samples. Total RNA (0.5 μg) was processed using the TruSeq RNA Sample Preparation Kit protocol (Illumina) according to the manufacture instructuctions . Libraries were evaluated by Qubit and Bioanalyzer. Sequencing libraries were constructed with barcodes to allow multiplexing of eight samples on one lane. The sequencing was run on an Illumina HiSeq 2000 instrument.
GEO Accession
GSM2127900