title: GSM1412737: HV+S+; Mus musculus; OTHER
library_construction_protocol: To isolate nuclei, cells were re-suspended in 5 ml LYSO-A buffer (35 mM Tris-Cl, pH7.5, 150 mM sucrose, 80 mM KCl, 5 mM MgCl2, 0.5 mM DTT) and centrifuged for 5 min at 1000 g. They were then re-suspended in 5 ml LYSO-A buffer containing 0.05% Tween-20. Cells were incubated on ice for 1-5 min, with constant checking to ensure that 90% of cells were permeabilised, using a haemocytometer and tryphan blue (Life Technologies). Cells were washed in 5 ml LYSO-A buffer and transferred to siliconized tubes (Eppendorf) in 1 ml LYSO-A. Cells were centrifuged at 900 g for 5 min and re-suspended in 500 μl F80 buffer (50 mM Tris, pH8.3, 40% glycerol, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT). Cells were counted and then re-suspended at 5 x 106 cells/100 μl before being snap-frozen in liquid nitrogen in aliquots of 100 μl. All steps were carried out at 4°C and using wide-bore pipette tips to minimize the loss of sample. GRO-seq libraries were prepared as in Core et al. (Core, Waterfall, and Lis, 2008), with the following modifications.  Trizol (invitrogen) was used to stop the reaction instead of DNase I and proteinase K treatment.  The RNA was further extracted once with acid phenol:chloroform, and once with chloroform before precipitating with 2.5 volumes of -20oC ethanol.  Bead binding buffers all contained 4units/ml of SUPERaseIN (ambion) and the following buffers were slightly modified.  Bead blocking buffer: 0.25X SSPE, 1mM EDTA, 0.05% tween, 0.1% PVP, and 1mg/ml ultrapure BSA (Ambion); Binding buffer: 0.25XSSPE, 37.5mM NaCl, 1mM EDTA, 0.05% tween; Low salt wash buffer: 0.2X SSPE, 1mM EDTA, 0.05% Tween.  High Salt wash buffer: 0.25% SSPE, 137.5mM NaCl, 1mM EDTA, 0.05% Tween.  The end repair steps were modified as follows.  Pelleted RNA from the first bead binding was resuspended in 20ul, and heated to 70oC for 5min, followed by incubation on ice for 2min.  1.5ul tobacco acid pyrophosphatase (TAP) buffer, 4.5ul water, 1 ul SUPERaseIn, and 1.5ul TAP (Epicentre) were then added and the reaction incubated at 37oC for 1.5 hours. 1ul 300mM MgCl2 and 1ul T4 polynucleotide Kinase (PNK) were added to the reaction for an additional protocols l 30 min. for phosphorylating the 5’-ends, 20ul T4 PNK buffer, 2ul 100mM ATP, 145ul water, 1ul SUPERaseIn, and an additional 2ul of PNK were added for 30 min at 37oC.  The reaction was then stopped by addition of 20mM EDTA followed by acid phenol extraction and precipitation.
attributes.tag: GEO Accession
attributes.value: GSM1412737