GSM1634336: RNA-Seq Undifferentiated EpSC shDNMT3B Replicate 2; Homo sapiens; RNA-Seq
RNA was extracted using TRIZOL (LifeTechnologies) and cleaned up through RNA binding affinity columns (Quiagen) following manufacturer instructions The library from total RNA from three biological replicates was prepared using the TruSeq®Stranded Total Sample Preparation kit (Illumina Inc.,) according to manufacturer's protocol. Briefly, rRNA was depleted from 0.5 ug of total RNA using the Ribo-Zero Gold Kit followed by fragmentation by divalent cations at elevated temperature resulting into fragments of 80-450nt, with the major peak at 160nt. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis, performed in the presence of dUTP instead of dTTP. Blunt-ended double stranded cDNA was 3´adenylated and the 3´-“T” nucleotide at the Illumina indexed adapters was used for the adapter ligation. The ligation product was amplified with 15 cycles of PCR. Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end mode with the read length 2x76bp. We generated minimally 137 million paired end reads for each sample run in one sequencing lane on HiSeq2000 (Illumina, Inc) following the manufacturer's protocol. Images analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA.
GEO Accession
GSM1634336