GSM5745856: Parp1KO_72hr_RNAseq_Rep1; Mus musculus; RNA-Seq
total RNA was extracted from mESCs using RNeasy kit (Qiagen, 74136), The integrity of the RNA was assessed and verified using an Experion Automated Electrophoresis System (Bio-Rad) before mRNA-seq libraries were prepared using methods described previously. polyA+ RNA was enriched using Dynabeads oligo(dT)25 (Invitrogen), heat fragmented, and reverse transcribed using random hexamers in the presence of dNTPs. Second strand cDNA synthesis was performed with dNTPs, but replacing dTTP with dUTP. After end-repair, dA-tailing, ligation to adaptors containing barcode sequences, and size selection using AMPure beads (Agencourt) for an insert length size of 150bp. The synthesized second-strand was digested using uracil DNA glycosylase (Enzymatics). A final PCR reaction was performed using Phusion high-fidelity DNA polymerase (NEB)