GSM3595482: 19_9_11_p70_GiWi_d30_Lac; Homo sapiens; RNA-Seq
Cell samples were collected using 0.25% trypsin-EDTA (Invitrogen) to remove the cells from cell culture plates. Total RNA was purified using QIAGEN RNeasy® Mini kit. Possible genomic DNA contamination was removed by RNase-Free DNase Set (QIAGEN) with the RNeasy columns. Total RNA was qualified with the Life Technologies Qubit fluourometer (Q32857) and Agilent Bioanalyzer (G2940CA). To prepare cDNA, the Ligation Mediated RNA sequencing library protocol was performed (Hou Z. et. al. Sci Rep 2015).  Briefly, mRNA was isolated from purified 100 ng total RNA using oligo-dT beads (NEB). Isolated mRNA was fragmented in reverse transcription buffer and reverse transcribed with SmartScribe reverse transcriptase (Clontech).   RNase A and RNase H treatments were then performed to remove RNA. The 5' adapter was ligated to the cDNA using RNA ligase 1 (NEB) and purified using Ampure XP Beads (Beckman).  The cDNA was then amplified with 18 cycles of PCR.  The indexed cDNA libraries were normalized and pooled at thirty samples per lane and sequenced on an Illumina HiSeq 2500 with a single 51bp read and a 10bp index read.
GEO Accession
GSM3595482