title: GSM7247477: Father GRO-seq repBss  (repB technical rep); Homo sapiens; OTHER
library_construction_protocol: Nuclie were preped from the cells.  RNA was isolated from the cells via Trizol extraction.    Wash solutions for Anti-BrdU-beads were prepared ahead of time;  Binding buffer (0.25X SSPE, 37.5 mM NaCl, 0.05\% Tween, 1mM EDTA pH 8, 0.2\% SuperRNAseIN), Low Salt Wash Buffer (0.25X SSPE, 0.05\% Tween, 1mM EDTA pH 8, 0.2\% SuperRNAseIN), High Salt Wash Buffer(0.25X SSPE, 137.5mM NaCl, 0.05\% Tween, 1 mM EDTA pH 8, 0.2\% SuperRNAseIN), TET Buffer(10 mM Tris-Cl pH 8.0,  0.05\% Tween, 1 mM EDTA pH 8.0, 0.2\% SuperRNAseIN), Elution Buffer (150 mM NaCl, 50 mM Tris-Cl pH 7.5, 20 mM DTT, 0.1\% SDS, 1 mM EDTA,  0.2\% SuperRNAseIN).      Anti-BrdU beads (Santa Cruz, sc-32323-ac) were prepared by washing and blocking. Per sample, 60µl were washed twice in 500 $\mu$l binding buffer. beads were blocked in 500 $\mu$l blocking buffer(1X binding buffer, 0.1\% PVP, 1 $\mu$g/mL BSA UltraPure, 0.002 more superRNAseIN). Beads were then resuspended in 450 $\mu$l binding buffer.    Run-on reactions were performed as in \cite{Core2008}. In brief, ice-cold isolated nuclei (100 $\mu$L) were added to 37$^\circ$C 100 $\mu$L reaction buffer (Final Concentration: 5 mM Tris-Cl pH 8.0, 2.5 mM MgCl$_2$, 0.5 mM DTT, 150 mM KCl, 10 units of SUPERase In, 0.5\% sarkosyl, 500 $\mu$M rATP, rGTP, and Bromo-UTP, 2 $\mu$M rCTP). The reaction was allowed to proceed for 10 min at 37$^\circ$C, followed by the addition of 1 $\mu$L of Trizol. RNA was extracted once with phenol-chloroform, washed once with chloroform, and precipitated with 3 volumes of ice-cold Etrisomy21ol and 1-2 $\mu$L GlycoBlue. The pellet was washed in 75\% Etrisomy21ol before resuspending in 18 $\mu$L of DEPC-treated water.    Libraries were prepared similar to \cite{Hunter2022}. In brief, RNA was treated with 2 $\mu$l NEB Fragmentation Buffer at 94 degrees for 5 min.  The RNA was then buffer exchanged via BioRad P-30 (or a G-25) column per the manufacturer's protocol. Next, 2 $\mu$l DNaseI and 5 $\mu$l of 10X RQ1 DNase buffer and water were added to create a 1X final concentration of the buffer. After incubation at 37$^\circ$C for 10min, 5 $\mu$l DNAse stop solution was added, and the reaction was placed at 65$^\circ$C for 5 min. As prepared above, the beads in 450 $\mu$l binding buffer was immediately added to this mixture.     Fragmented nascent RNA was purified using Anti-BrdU beads via washing with 500 $\mu$l for 1 min with each solution (binding buffer, low salt buffer, high salt buffer, TET buffer). Between washes, beads were spun down per manufacturer instructions. RNA was eluted from the bead via soaking the beads in 125 $\mu$l  elution buffer at 42$^\circ$C, 10 min 2 times. The beads were then added to acid phenol-chloroform, as was the elute. The RNA was washed with chloroform and precipitated with 5M NaCl and 3x the Etrisomy21ol column.  The second round of Anti-BrdU bead binding and extraction enriched BrdU-labeled products was completed as above. NEBNext Ultra II RNA was used to transform the nascent RNA into an RNA-seq library. The product was amplified 15 $\pm$ three cycles and products >150 bp (insert > 70 bp) were size selected with 1X AMPure XP beads (Beckman) before being sequenced.