GSM1370477: 4sU_RNA-seq exp2, 3T3-L1 4h; Mus musculus; RNA-Seq
4sU-RNA-seq was performed essentially as described previously (Rabani et al., 2011, Nature biotechnology 29, 436-442). Briefly, cells were incubated with 400 μM 4-thiouridine (4sU) for 30 min and then harvested in TRIzol (Invitrogen). RNA was then purified according to the manufacturer’s instructions. Purified RNA was biotinylated and pulled down using streptavidin beads. Enriched RNA was then purified and subjected to standard mRNA sample preparation for sequencing and sequenced on the Illumina platform according to the manufacturer’s instructions. ChIP-seq: Chromatin was prepared from 150 mm culture dishes first by crosslinking in 1% formaldehyde in PBS (10 minutes, RT). Cross-linking was stopped by adding glycine to a final concentration of 0.125 M (10 minutes, RT). The cells were washed twice in ice-cold PBS, and harvested in ice-cold lysis buffer (0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris pH=8) and sonicated at high setting in a Bioruptor-Twin (Diagenode) at a volume of 1.5 ml in 15-mL tubes for 40 cycles of 30 seconds on and 30 seconds off. The chromatin IPs were performed as described in (Nielsen R, Mandrup S, 2014 Genome-Wide Profiling of Transcription Factor Binding and Epigenetic Marks in Adipocytes by ChIP-seq. Methods in Enzymology 2014, Vol. 537, pp. 261-279). Chromatin used for ChIP-seq of p300 and MED1 was also cross-linked in 2 mM disuccinimidyl glutarate (DSG) for 45 minutes at RT prior to cross-linking by formaldehyde. RNA-seq: RNA was purified using TRIzol (Invitrogen) according to the manufacturer’s instructions.
GEO Accession
GSM1370477