title: Illumina HiSeq 2000 paired end sequencing; RIP-seq to identify transcripts interacting with pluripotency factors in mouse embryonic stem cells
design: RIP-seq to identify transcripts interacting with pluripotency factors in mouse embryonic stem cells
library_construction_protocol: J1 cells expressing the biotin ligase BirA and Sox2, Nanog or Oct4 flanked by a peptide amenable to biotinylation by BirA, as well as control J1 cells only expressing BirA, were kindly provided by Dr. Stuart H. Orkin (Dana Farber, Harvard Medical School). E14 cells were kindly provided by Dr. José Silva (Cambridge University). All cell lines were grown on 0.1% gelatin coated plates and maintained in ES media consisting of Glasgow Minimum Essential Medium (GMEM) supplemented with 10% fetal calf serum for ESCs (Biosera, Boussen, France), 0.1 mmol/L non-essential amino acids, 2 mmol/l L-Glutamine, 1 mmol/l sodium pyruvate, 0.1 mmol/l beta-mercaptoethanol, 1x penicillin/streptomycin and 106 units/L LIF (ESGRO, MilliporeCorp., Billerica, MA, USA). All incubations were performed in low-bind RNase-free tubes. 50 million cells/IP were fixed with 1% formaldehyde (Sigma F8775) for 10 min at room temperature, quenched with Glycine stop solution (Active Motif, ChIP-IT Enzymatic Kit, catalogue number 53006) and lysed. The lysate was either sonicated at high frequency (H 20W) with the 30 s ON/30 s OFF setting for 20 min in BioRuptor or processed for nuclei isolation and enzymatic digestion (Active Motif, ChIP-IT Enzymatic Kit, catalogue number 53006). Sheared chromatin was pre-cleared with Protein G Dynabeads for 1 h at 4°C with gentle rotation. Immunoprecipitation of biotinylated Sox2/Oct4/Nanog was performed with 50 l MyOne streptavidin Dynabeads T1 for 3 h or overnight followed by washing with FA1000, LiCl and TES buffers. For endogenous RIPs, the following antibodies were used: mouse-FLAG M2 (Sigma), Sox2 (α-Sox2, raised in goat, Y-17, Santa Cruz, sc-17320), Nanog Antibody AbVantage Pack (Bethyl, A310-110A), Mouse KLF4 Affinity Purified Polyclonal Ab, Goat IgG (AF3158, R&D Systems), Oct-3/4 (N-19) X, Polyclonal Antibody (sc-8628-X, Santa Cruz) and Suz12 (Abcam, ab12073). RNA was eluted after reverse-crosslinking (65°C for 1 h with 1,000 rpm rotation), Qiazol was added to IP samples and 10% input samples (collected before the IPS) and RNA was extracted using the Qiagen miRNAeasy kit. The TruSeq directional small RNA kit (Illumina RS-200-0012) was used. In order to capture both long and short RNAs, RNA was fragmented (Ambion AM8740) prior to sample preparation. Total RNA was depleted from ribosomal RNA by treatment with Ribo-Zero rRNA removal kit (RZH1086).
attributes.tag: Experimental Factor: genotype
attributes.value: birA, bioSox2
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: NA
attributes.tag: Experimental Factor: IP time
attributes.value: NA