GSM4551320: PROseq_CD34_BCLKO_72hr_rep1; Homo sapiens; OTHER
Ten million CD34+ cells undergoing erythroid differentiation were harvested on ice, and washed with cold PBS. Cells were resuspended in 2 mL cold Buffer W (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 0.5 mM DTT, 10% glycerol, 0.5% BSA) using wide-bore P1000 tip and transferred to a 2 mL low binding tube, and strained through a 35 micron nylon mesh cell strainer. Immediately add 18 mL Chilled Buffer P (Buffer W supplemented with 0.05% Igepal CA-630) to cell suspension and mix gently for 2 min. Cells were centrifuged at 400xg for 8 min and resuspended in 1 mL Buffer F (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT, 0.5% BSA), and centrifuged again at 400xg for 8 min. The pellets were resuspended in 400 uL Buffer F, counted using a hemocytometer to ensure that >95% cells were permeabilized. The cell density was adjusted to 1 x 106/100 uL, and the suspension was snap-frozen and stored at -80°C.An aliquot of frozen cells were thawed on ice and counted. For each sample, take 1 million cells and spike in 50,000 permeabilized Drosophila S2 cells, and add the same volume of 2x Nuclear Run-On buffer (10 mM Tris-HCl pH 8.0, 300 mM KCl, 1% Sarkosyl, 5 mM MgCl2, 1 mM DTT, 200 uM biotin-11-A/C/G/UTP (Perkin-Elmer), 0.8 u/mL SUPERaseIN inhibitor [Ambion]) and then incubate at 30°C for 5 min. Total RNA was isolated and fragmented by base hydrolysis with 0.25 N NaOH on ince for 10 min, and neutralized with 1x volume of Tris-HCl pH 6.8. Biotinylated RNA was bound to 30 uL Streptavidin M-280 beads (Thermo Fisher Scientific) in Binding Buffer (300 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100). Beads were washed twice with High salt buffer (2 M NaCl, 50 mM Tris- HCl pH 7.4, 0.5% Triton X-100), twice in Binding Buffer, and twice in Low salt buffer (5 mM Tris-HCl pH 7.4, 0.1% Triton X-100). Bound RNA was extracted using Trizol (Invitrogen), precipitated using ethanol and dissolved in H2O. Nascent RNA was ligated with 10 pmol of 3' RNA adaptor (5'p-rNrNrNrNrNrNrGrArUrCrGrUrCrGrGrArCrUrGrUrArGrArArCrUrCrUrGrArArC-/3'InvdT/) and T4 RNA ligase I (NEB) at 20°C for 2hrs. Ligated RNA was enriched with another round of streptavidin beads purification with one additional round of washing with 1x ThermoPol Buffer (NEB). RNA products were treated with RNA 5' Pyrophosphohydrolase (RppH, NEB) at 37°C for 30 min followed by one wash of High, Low and T4 PNK Buffer. 5' ends were repaired with Polynucleotide Kinase (PNK, NEB) at 37°C for 30 min. RNA was ligated to 5' RNA adaptor 5'-rCrCrUrUrGrGrCrArCrCrCrGrArGrArArUrUrCrCrA-3' with T4 RNA ligate I (NEB) for 2 hrs at 20 °C, and enriched with streptavidin beads with additional wash using 1x SuperScript IV buffer (Thermo Fisher Scientific) and reverse transcribed using 25 pmol RT primer AATGATACGGCGACCACC GAGATCTACACGTTCAGAGTTCTACAGTCCGA. 12.5 pmol RPI index primers and RT pimer were added to the RT product for library amplification.
GEO Accession
GSM4551320