GSM1199552: RNA-seq of mRNA from  Mouse Ter119+ erythroid  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 Paired sequencing library was generated using the standard Illumina prtocol. Total RNA quantity and integrity were assessed, using Quant-IT RiboGreen RNA Assay Kit (Invitrogen, Carlsbad, CA, USA) and Agilent Tapestation 2200 R6K. mRNA enrichment was achieved by processing 5µg 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 with two Ampure Bead bindings (firstly, 1:0.7x volume and secondly, the supernatant from the first bind was taken for a 1:1.7x volume clean-up).   The following custom primers (25 µM each) were used for the PCR enrichment step:  Multiplex PCR primer 1.05'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'  Index primer5'-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'   Indices were according to the eight bases tags developed by WTCHG (manuscript in preparation).  For libraries that were run as pools:  Amplified libraries were analysed for size distribution using the Agilent Tapestation 2200 D1K. Libraries were quantified by quantitative RT-PCR using Agilent qPCR Library Quantification Kit and a MX3005P instrument (Agilent) and relative volumes were pooled accordingly. Finally, a second quantitative RT-PCR 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 paired end read on a HiSeq2000 according to Illumina specifications.  For non-pooled libraries:  Amplified libraries were analysed for size distribution using the Agilent Tapestation 2200 D1K. Quantitative RT-PCR was performed to measure the relative concentration of the library compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing. Sequencing was performed as 50bp Paired End read on a HiSeq2000 according to Illumina specifications.
GEO Accession
GSM1199552