GSM1518098: U1D_24h; Mus musculus; RNA-Seq
For nuclear RNA extraction, cells collected by a scrapper were suspended in cell lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 0.5% NP40, 1 mM DTT), followed by vortexing for 10 sec and incubation on ice for 10 min. After centrifugation of the lysate at 500 x g for 5 min at 4oC, the pellet was re-suspended in the cell lysis buffer for nuclear RNA extraction. Both total and nuclear RNAs were extracted using Trizol (Invitrogen) according to manufacturer's protocol. RNA quality was analyzed in an Agilent Bioanalyzer using the RNA pico600 kit before processing for deep sequencing. The 3'READS method used in this study was previously described (Hoque et al., 2014). Briefly, 25 μg of total RNA was used for each sample, and poly(A)+ RNA was selected using oligo d(T)25 magnetic beads (NEB), followed by on-bead fragmentation using RNase III (NEB). Poly(A)+ RNA fragments were then selected using the chimeric U5 and T45 (CU5T45) oligo conjugated on streptavidin beads, followed by RNase H digestion (NEB). Eluted RNA fragments were ligated with 5' and 3' adapters, followed by RT and PCR (15x) to obtain cDNA libraries for sequencing on the Illumina platform.
GEO
GSM1518098
GEO Accession
GSM1518098