GSM2415883: Lib153OverExpressTRUB2rep1_input; Homo sapiens; RNA-Seq
Enrichment of polyadenylated RNA (polyA+ RNA) from total RNA was performed using one round of enrichment using Oligo(dT) dynabeads (Invitrogen) according to the manufacturer’s protocol. CMC-treatment was performed essentially as described in (Schwartz et al, Cell, 2014). The mRNA was chemically fragmented into ~80-150 nt-long fragments by performing a short fragmentation (30 seconds) using RNA fragmentation reagent and stop solution (Ambion). RNA was subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) or SuperScript III (Life Technologies), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9-12 cycles in a PCR reaction. Libraries were sequenced on Illumina HiSeq 2500 platforms generating paired end reads.
GEO Accession
GSM2415883