GSM1518021: WT ESCs 0h after Actinomycin treatment; Mus musculus; RNA-Seq
Total RNA was fragmented into average size of 300 nucleotides by incubation of the RNA in 95oC for 4:30 minutes (NEBNext Magnesium RNA Fragmentation Module). The 3’ polyadenylated fragments were enriched by selection on poly dT beads (Dynabeads Invitrogen). Strand specific cDNA was synthesized using a poly T-VN oligo (18 T) and Affinity Script RT enzyme (Agilent). Double strand DNA was obtained using Second strand synthesis kit (NEBNext second strand synthesis module). DNA was purified by adding paramagnetic SPRI beads (Agencourt AMPure XP, Beckman Coulter) pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a 96-well magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction. DNA ends are first repaired by T4 polymerase (NEBNext), next, T4 polynucleotide kinase (NEB) adds a phosphate group at the 5′ ends. An adenosine base is then added to the blunt-ended fragments, using Klenow enzyme (NEB), and a barcode Illumina compatible adaptor (IDT) was ligated to each fragment using T4 quick ligase (NEB). DNA fragments were amplified by 12 cycles of PCR (Kapa HiFi) using specific primers (IDT) to the ligated adaptors. Library quantity was determined by Qubit Fluorometric Quantitiation (Life Technologies) and the quality of each library was analyzed by Tapestation (Agilent).
GEO
GSM1518021
GEO Accession
GSM1518021