GSM5658226: Gro-seq-siCTL-E2-MCF7; Homo sapiens; OTHER
MCF7 cells were washed 3 times with cold phosphate buffered saline (PBS) buffer and then incubated in swelling buffer (10 mM Tris-Cl pH 7.5, 2 mM MgCl2, 3 mM CaCl2) for 5mins on ice and harvested. Cells were first re-suspended and lysed in lysis buffer (swelling buffer with 0.5% IGEPAL and 10% glycerol). The resultant nuclei were washed once again with 10 mL lysis buffer and then re-suspended in 100 μL of freezing buffer (50 mM Tris-Cl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). For run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 20 units of SUPERase In, 1% sarkosyl, 500 μM ATP, GTP, and Br-UTP, 2 μM CTP) and incubated for 5 mins at 30°C. The nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent following manufacturer's instructions. NRO-RNA was then subjected to base hydrolysis on ice for 40 mins and followed by treatment with DNase I and antarctic phosphatase. To purify the Br-UTP labeled nascent RNA, the NRO-RNA was immunoprecipitated with an anti-BrdU agarose beads in binding buffer (0.5 X SSPE, 1 mM EDTA, 0.05% tween-20) for 1hr at 4°C while rotating.  To repair the end, the immunoprecipitated BrU-RNA was re-suspended in 50μL reaction (43 μL DEPC water, 5.2 μL T4 PNK buffer, 1 μL SUPERase In and 1 μL T4 PNK) and incubated at 37 °C for 1hr. RNA was then extracted, precipitated using acidic phenol-chloroform and subjected to poly-A tailing reaction by using poly-A polymerase for 30 mins at 37 °C. Subsequently, reverse transcription was performed using oNTI223- primers (5'- pGATCGTCGGACTGTAGAACTCT; CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3') where the p indicates 5' phosphorylation, ';' indicates the abasic dSpacer furan and VN indicates degenerate nucleotides. Specifically, tailed RNA (8.0 μL) was subjected to reverse transcription using superscript III, after which the cDNA products were separated on a 10% polyacrylamide TBE-urea gel. The extended first-strand product (100-500 bp) was excised and recovered by gel extraction. After that, the first-strand cDNA was circularized by CircLigase and re-linearized by Ape1. Re-linearized single strand cDNA (sscDNA) was separated in a 10% polyacrylamide TBE gel as described above and the product of needed size was excised (~170-400 bp) for gel extraction. Finally, sscDNA template was amplified by PCR using the Phusion High-Fidelity enzyme according to the manufacturer's instructions. Primers oNTI200 (5'-CAAGCAGAAGACGGCATA-3') and oNTI201 (5'- AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG-3') were used to generate DNAs for deep sequencing.