GSM4633449: NEBNext_Ultra_II_RNA_UD_mESC_replicate_3; Mus musculus; RNA-Seq
Total RNA was extracted from cells with TRIzol (Invitrogen), according to the manufacturer's instructions. The resulting total RNA was treated with DNase I (NEB) to avoid genomic DNA contamination. Phenol/chloroform extraction and ethanol precipitation were then performed to purify and concentrate total RNA. For mRNA isolation, two successive rounds of poly(A)+ selection were performed using oligo(dT)25 dynabeads (Invitrogen). Reverse transcription was carried out using Superscript IV reverse transcriptase (Invitrogen) and then tagmentation reaction was performed with Tn5 transposase (Vazyme). Strand extension reaction was performed by directly adding Bst 3.0 DNA Polymerase and 1X NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB) to tagmentation products and incubating at 72°C for 15 min, followed by Bst 3.0 DNA Polymerase inactivation at 95°C for 5 min. Next, 0.2 μM indexed primers were added to perform enrichment PCR.The library was purified twice using 1X Agencourt AMPure XP beads (Beckman Coulter) and sequenced on the Illumina Hiseq X10 platform which generated 2 x 150 bp of paired-end raw reads.
GEO Accession
GSM4633449