GSM5034226: HCT116-NUP93-mAC, PRO-Seq, IAA, biological replicate 1; Homo sapiens; OTHER
about 10 million cell nuclei were extracted on ice. They were incubated in the nuclear run-on buffer at 37°C for 5minutes in the presence of 500μM of ATP and GTP together with 50μM of biotin-11-CTP and biotin-11-UTP. Total RNAs were extracted using TRIzol and were fragmented using NaOH hydrolysis on ice for 10 minutes. The fragmented biotin labeled nuclear Run-on RNAs (NRO RNAs) were purified using the Dynabeads® MyOne™ Streptavidin C1 (Thermo Fisher 65002). The NRO RNAs were further purified twice using TRIzol beads. For library making, the NRO RNAs were end-repaired by a fast Alkaline Phosphatase (Thermo Fisher) and by T4 PNK (NEB), cleaned up using Dynabeads MyOne Silane (37002D), and were then ligated to a HPLC purified 3'end RNA linker: RiL19 /5phos/rArGrArUrCrGrGrArArGrArGrCrGrUrCrGrUrG/3SpC3/. The ligated RNA was reverse transcribed using AffinityScript first strand cDNA synthesis kit (Agilent) by using AR17 primer (5'- ACACGACGCTCTTCCGA - 3').  The cDNA was then cleaned up and ligated with a DNA linker carrying 10 nucleotides unique molecular index (UMI), rand10-3Tr3 (/5Phos/NNNNNNNNNNAGATCGGAAGAGCACACGTCTG/3SpC3/). This cDNA with both ends carrying ligated adaptors was cleaned up by Dynabeads MyOne Silane (37002D), and PCR was conducted to test the library abundance. Final PCR primers for library amplification were based on PCR_F_D501: 5'- AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCTCTTCCGATCT -3' and one of the indexed 3'end primers PCR_Rev_p7x: 5'- CAAGCAGAAGACGGCATACGAGAT XXXXXX GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC  -3' (where XXXXXX indicates 6-nucleotide index sequence), and the library was made using NEB Q5 2x PCR master mix by around 9-13 cycles. Final library was sized-selected using TBE 10% PAGE gels with a range of ~170-400nt, or by dual size selection using KAPApure beads (0.55X and 0.9X).
GEO Accession
GSM5034226