title: GSM3537073: mESC-control-1; Mus musculus; RNA-Seq
library_construction_protocol: Cell culture: ESCs were maintained in strandard ES medium supplemented with 15% heat-inactivated FCS, 1% of nucleoside mix (100 × stock, Millipore), Penicillin-Streptomycin Solution (100 × stock, Life Technologies), 2 mM Glutamax (100 × Life Technology), 0.1 mM non-essential amino acid, 0.1 mM 2-mercaptoethanol and supplied with 1000 U/ml recombinant leukemia inhibitory factor (LIF, millipore). RNA-seq: Total RNA of RNA-Seq were extracted by TRIzol reagent according to manufacturer's instruction. mRNA were further isolated by Dynabeads mRNA purification kit (Life Technologies) according to manufacturer's instruction. ChIP-seq: BirA- or FLAG-Biotin-ESCs were cross-linked with 1% formaldehyde for 10 min at room temperature and quenched with glycine to a final concentration of 0.125 M for another 5min. Chromatin was sonicated, cleared by centrifugation, and incubated overnight at 4℃ with Dynabeads M280 streptavidin. followed by stringent washes and elution. Eluates were reverse cross-linked overnight at 65℃ and deproteinated with proteinase K at 56℃ for 30 min. DNA was purified using DNA purification kit (Qiagen). CLIP and TAP-seq: The CLIP method was performed as described before with the specific modifications below. TAP method was developed by our lab sharing most of the same steps with CLIP, but including tandom purification and avoiding SDS-PAGE separation. About 107 cells of FbioDDX18 ESCs grown on one 15-cm plate were harvested and resuspended with 5 ml cold PBS. The cells were plated back to the plate and crosslinked with 600 mJ/cm2 of 254 nm UV light. The nucleuses were prepared by incubated the cells in hypotonic buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 0.2% NP-40, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1/500 proteinase inhibitor cocktail, 400 U/ml RNase Inhibitor) for 4 minutes and the cells were lysed by a 26 5/8 Gauge needle and briefly sonicated. Incubate the cells for 30mins on a rotator at 4℃. Treat the lysate with 30 µl RQ1 DNase at 37℃ for 10mins. The insoluble pellet was spun down and the supernatant was harvested. About 40 µl pre-equilibrated FLAG M2 resins (Sigma) were used for each purification. The FLAG resins were incubated with lysate overnight at 4 °C and washed twice with cold CLIP lysis buffer and two more times with high salt wash buffer (5XPBS, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). For TAP experiments, the proteins were eluted with 200 ng/ml 3xFLAG peptide dissolved in CLIP lysis buffer (sigma) for three times. The eluents were pooled together and incubated with 60 µl pre-equilibrated streptavidin (M-280, Invitrogen) beads for three hours. The samples were washed twice with SDS-urea wash buffer (50 mM Tris pH 7.4, 2% SDS, 1 M urea) and twice with SDS-high salt wash buffer (5XPBS, 0.5% NP-40, 0.5% SDS) at room temperature. The protein-RNA complex bound beads were washed four times with PNK buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.5% NP-40). Then the RNAs were partially digested with MNase (NEB, 1:105 diluted) at 37 °C for 10 min (mixed with an Eppendorf Thermomixer at 1,200 rpm for 5 second per 30 seconds). The reaction was stopped by PNK-EGTA buffer (PNK buffer with 2 mM EGTA). Then the beads were washed twice with SDS-urea wash buffer and twice with PNK buffer. To dephosphorylate the RNA, the beads were treated with CIP (NEB) for 10 min at 37 °C (mixed with an Eppendorf Thermomixer at 1,200 rpm for 5 seconds per 30 seconds). After two more washes with PNK buffer, the pre-adenylated 3' linker (rAppAGATCGGAAGAGCACACGTCT-NH2, Takara) was ligated with truncated T4 RNA ligase 2 (NEB) at 16 °C overnight. The non-ligated 3' linker was washed away with two washes of CLIP lysis buffer and two washes of PNK buffer. The RNAs were then phosphorylated by T4 PNK (NEB) for 10 min at 37 °C. Then for CLIP experiments, the RNA was subjected to SDS–PAGE separation and transfer, autoradiograph, ribonucleoprotein isolation, proteinase K treatment, and overnight RNA precipitation. For TAP experiments, the samples were treated with Proteinase K (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% SDS, and 20 µg Proteinase K) for 30 min at 37 °C to elute the protein-RNA complex. Then the RNAs were purified and ligated with 5' RNA adaptor (GUUCAGAGUUCUACAGUCCGACGAUC, TAKARA) by T4 RNA ligase (Ambion). Then the RNAs were reverse transcribed by Superscript III with RT primer (AGACGTGTGCTCTTCCGATCT, Takara) and amplified for 20 cycles with Q5 DNA polymerase (NEB) NEBNext Ultra RNA/DNA Library Prep Kit for Illumina
identifiers.namespace: GEO
identifiers.id: GSM3537073