title: GSM7100824: CN,fastGRO_CON_1; Mus musculus; OTHER
library_construction_protocol: DIV13 primary cortical neurons (~20M cells per replicate) were washed twice with ice-cold PBS and collected in a 15 mL conical tube. Cells were incubated in 10 mL swelling buffer (10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 2 U/ml Superase-In (Invitrogen)) for 5 minutes on ice, washed with swelling buffer + 10% glycerol, and resuspended in 10 mL lysis buffer (10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 10% glycerol, 1% IGEPAL, 2 U/ml Superase-In) to isolate nuclei. Isolated nuclei were washed twice with lysis buffer and resuspended in 100 µl freezing buffer (50 mM Tris-HCl pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol). An equal volume of pre-warmed, 2X nuclear run-on reaction buffer (10 mM Tris-HCl pH 8, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM 4-thio-UTP (Trilink), 2 µM CTP, 200 U/ml Superase-in, 1% sarkosyl) was added, and the reaction was incubated for 7 minutes at 30°C. Nuclear run-on (NRO) RNA was extracted with TRIzol LS reagent (Invitrogen) following the manufacturer's instructions and precipitated with ethanol. Precipitated NRO-RNA was resuspended in 100 µl dH2O, and concentration was determined using a Qubit 4.0 fluorimeter (Thermo). RNA (120-150 µg) was transferred to a new tube and 5 % - 10 % spike-in RNA derived from S2 cells was added. RNA was then fragmented using a bioruptor (Diagenode; high power, 30 seconds on/30 seconds off for 3 cycles) Fragmentation efficiency was analyzed by running fragmented and unfragmented RNA on an Agilent 4200 TapeStation instrument using High Sensitivity RNA ScreenTape following manufacturer's instructions. Fragmented RNA was incubated in biotinylation solution (20 mM Tris pH 7.5, 2 mM EDTA, 40% dimethylformamide, 200 µg/ml EZ-link PDP Biotin (Thermo)) for 2 hours at 25°C with rotation (800 rpm) in a dark room then purified via ethanol precipitation and resuspended in dH2O. M280 streptavidin Dynabeads (Invitrogen) were washed twice with 2 volumes of freshly prepared wash buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1M NaCl, 0.1% Tween-20) and resuspended in 1 volume of wash buffer. Thereafter, biotinylated RNA was affinity purified via incubation with 100 µl washed Dynabeads for 15 minutes at 4°C. After binding, Dynabeads were washed three times with wash buffer pre-warmed at 65°C, and three times with wash buffer at room temperature. RNA was eluted with 100 mM DTT buffer and purified with RNA Clean and Purification kit (Zymo Research) with in-column DNase reaction to eliminate traces of genomic DNA.