GSM1573195: GROseq_siTbx19_hs10l3_4; Mus musculus; RNA-Seq
GRO-seq experiments were performed as previously reported. Briefly, 20 millions of AtT20 cells, treated with siRNAs for 2 days, were washed 3 times with cold PBS, and then incubated in swelling buffer (10mM Tris-HCl pH7.5, 2mM MgCl2, 3mM CaCl2) on ice for 5 min. Cells were lysed in 1ml of lysis buffer (swelling buffer plus 0.5% NP-40, 10% glycerol and 20 units of SUPERase-In) with 15 sec vortex at 800 rpm. The nuclei pellets were washed twice with 10ml lysis buffer and 1 ml of freezing buffer (50mM Tris-HCl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). The isolated nuclei were suspended in 100μl of freezing buffer. For the run-on assay, an equal volume of reaction buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 300mM KCl, 1% sarkosyl, 500μM ATP, GTP, and Br-UTP, 2μM CTP,  1mM DTT, 20 units of SUPERase-In) was added to the isolated nuclei, and incubated for 5 minute at 30°C. The nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Life Technologies). NRO-RNA was hydrolyzed to ~300-500 nucleotides by alkaline base on ice for 30 min and followed by treatment with DNase I and antarctic phosphatase. The fragmented NRO-RNA was pulled down with anti-BrdU argarose beads (Santa Cruz Biotechnology) in binding buffer (0.5XSSPE, 1mM EDTA, 0.05% tween) for 1-3 hours at 4°C with rotation. Subsequently, T4 PNK was used to repair the ends of the precipitated Br-UTP labeled RNA at 37°C for 1hour. The RNA was extracted and precipitated using acidic phenol-chloroform. The RNA fragments were subjected to poly-A tailing reaction by poly-A polymerase (NEB) for 30 minute at 37°C, and reverse transcribed using oNTI223 primer and superscript III RT kit (Life Technologies). The 100-500 bp cDNA products were isolated using 10% polyacrylamide TBE-urea gels. Next, the purified first-strand cDNA was circularized by CircLigase (Epicentre) and re-linearized by APE1 (NEB). ~120-320bp of re-linearized single strand cDNA (sscDNA) was isolated with a 10% polyacrylamide TBE gel. Finally, the purified sscDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB). The oligonucleotide primers oNTI200 and oNTI201 were used to generate DNA for deep sequencing. DNA was ligated to specific adaptors followed by deep sequencing on the Illumina GAII system according to the manufacturer’s instructions.
GEO
GSM1573195