GSM1156606
GSM1156606: RNA-seq of E14-TG2a.IV ES cells; Mus musculus; RNA-Seq
RNA was isolated with Tri-Reagent (Sigma). The RNA quality was assessed using Agilent Bioanalyser. Only RNA with overall RIN score >9 was used for further procedures. Subsequently, RNA was treated with DNAseI (DNA-free, Ambion) according to the manufacturer's protocol kit according to the protocol. Good quality and DNA-free RNA was subjected to polyA fractionation using PolyA selection kit (Promega) using the standard kit protocol.  The RNA fraction was precipitated overnight and resuspended with water. The quality of obtained RNA samples was assessed using PicoChip (Agilent). A single end sequencing library was generated using the standard Illumina prtocol. Samples were quantified using Nanodrop (Invitrogen) and quality controlled on a 6000 Nano Bioanalyzer chip (Agilent). mRNA enrichment was achieved by processing 6µg of total RNA using the Magnetic mRNA Isolation Kit from NEB (S1550S) with minor modifications.  Generation of double stranded cDNA and library construction were performed using NEBNext® mRNA Sample Prep Reagent Set 1 (E6100L) according to manufacturer specifications.  Upon ligation of Illumina Adapters (Multiplexing Sample Preparation Oliogonucleotide Kit) each library was size selected using a 2% E-Gel® EX (Invitrogen).  The following custom primers (25 µM each) were used for the PCR enrichment  step:  Multiplex PCR primer 1.0  5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC CGATCT-3'  Index primer  5'-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCAGAC GTGTGCTCTTCCGATCT-3'  Indices were according to the eight bases tags developed by WTCHG (manuscript in preparation).  The amplified library was purified using Ampure beads (Beckman/Agencourt) and size distribution determined using Agilent DNA 1000 Kit and a 2100 Bioanalyzer Instrument system (Agilent). Libraries were quantified by realtime using Agilent QPCR Library Quantification Kit and a MX3005P instrument (Agilent) and pooled accordingly. Finally, a second realtime was performed to measure the relative concentration of the pool compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing. . Sequencing was performed as 50bp single read on a GAIIx according to Illumina specifications using Cluster Generation Kit v4 and Sequencing Kit v4.
GEO
GSM1156606
GEO Accession
GSM1156606