title: GSM1480327: K562 PRO-Seq; Homo sapiens; OTHER
library_construction_protocol: Nuclei isolation: All steps performed and buffers kept at 4oC. 10 mls of cells were spun down at 700g in a swinging bucket rotor for 5 min. Cells were washed in 1X cold PBS. Cells were washed in 10ml of buffer W (10mM Tris-Cl, pH 7.5, 10mM KCl, 150mM sucrose, 5mM MgCl2, 0.5mM CaCl2, and 0.5 mM DTT). Cells were resuspended in 5mls of Buffer P (10mM Tris-Cl, pH 7.5, 10mM KCl, 250mM sucrose, 5mM MgCl2, 1mM EGTA, and 0.5 mM DTT, .05% tween-20 and/or .1% Igepal), and incubated 5 min. on ice. Permeabilized cells were washed twice in 5ml of buffer P, resuspended in 1ml of buffer P, and transferred to a 1.7ml eppendorf tube. Cells were pelleted at 1000 x g in a fixed angle rotor for 5min and then resuspended in 500 ml of buffer F (50 mM Tris-Cl pH 8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA, 0.5mM DTT). Cells were pelleted at 100g for 5 min, resuspended in 100 ml of buffer F, and then flash frozen in liquid nitrogen. PRO-seq libraries were prepared as described previously (Kwak et al., 2013). Briefly, 5×10^6 nuclei were added to 2 X Nuclear Run-On (NRO) reaction mixture (10 mM Tris-HCl pH 8.0, 300 mM KCl, 1% Sarkosyl, 5 mM MgCl2, 1 mM DTT, 0.375 mM each of biotin-11-A/C/G/UTP (Perkin-Elmer), 0.8 u/µl RNase inhibitor) and incubated for 3 min at 30˚C. Nascent RNA was extracted and fragmented by base hydrolysis in 0.2 N NaOH on ice for 10~12 min, and neutralized by adding 1 X volume of 1 M Tris-HCl pH 6.8. Fragmented nascent RNA was purified using streptavidin beads, ligated with reverse 3' RNA adapter (5'p-GAUCGUCGGACUG-UAGAACUCUGAAC-/3'InvdT/), and biotin-labeled products were enriched by another round of streptavidin bead binding and extraction. For 5' end repair, the RNA products were successively treated with tobacco acid pyrophosphatase (TAP, Epicentre) and polynucleotide kinase (PNK, NEB). 5' repaired RNA was ligated to reverse 5' RNA adaptor (5'-CCUUGGCACCCGAGAAUUCCA-3') before being further purified by the third round of streptavidin bead binding and extraction. RNA was reverse transcribed using 25 pmol Illumina RP1 primer (5'-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3'). The product was amplified 15 cycles and products greater than 120 bp (insert > 70 bp) were PAGE purified before being analyzed by Illumina HiSeq 2500 instrument.
attributes.tag: GEO Accession
attributes.value: GSM1480327