GSM3834596: Bcell_GROSeq_Spt5dep2_24474; Mus musculus; OTHER
Following cell membrane lysis with hypotonic buffer, nuclei were isolated via sucrose gradient density centrifugation. GROseq was performed as described (Core & Lis, 2008). Briefly, 10 million nuclei were used per reaction, and the run-on was performed for 5 minutes with NTPs with a  limiting concentration of P32-dCTP and dBrdUTP followed by extraction of RNA with Trizol reagent (Thermo Fisher, 15596026). An updated protocol was kindly provided by Dr. John Lis (Cornell University). After base hydrolysis and DNA end repair, Illumina compatible 3' RNA adapters were ligated to total RNA. After BrdU- incorporated RNA IP the 5' cap was removed and the 5' DNA end was repaired. Next, the 5' DNA Illumina comaptible adapters were ligated, and the library was reverse transcribed and amplified.
GEO Accession
GSM3834596