GSM3359500: oocyte_IV_RNA_seq; Danio rerio; RNA-Seq
Total RNA of oocytes were purified using RNeasy Mini kit (QIAGEN, 74104) as recommended protocols 5 ug RNA was DNase I (Fermentas, Cat # EN0521) treated at 37C for 1 h. RNA was then purified using AMPure beads. Poly-A tailed mRNA was collected using DynabeadsTM mRNA purification kit (Invitrogen, Cat # 61006). Purified RNA was fragmented with RNA Frag. Buffer (NEB, Cat # E6186A) at 95C for 5 min. Reaction was stopped and RNA was purified by AMPure beads. First stand cDNA was synthesized with a commercial kit using both oligo dT and random primers (Invitrogen, Cat # 18080-051). Second strand cDNA was synthesized with second strand synthesis buffer (Invitrogen, Cat # 10812014), MgCl2, DTT, dNTP, dUTP, RNase H (Fermentas, Cat # EN0202), E. coli DNA ligase (NEB, Cat # M0205S) and DNA polymerase I (NEB, Cat # M0209S). DNA was purified after 2 hrs incubation on thermomixer at 16C. Synthesized cDNA was subjected to library preparation. DNA was end-repaired, adenylated, and ligated to TruSeq sequencing adaptors. Libraries were then treated with UDG (AMPErase, Cat # N808-0096) at 37C for 1 hour followed by DNA amplification using Phusion HF DNA polymerase (NEB, Cat # M0530L). The amplified DNA was size selected using AMPure Beads for 200-500 bp DNA fragments.
GEO
GSM3359500