title: GSM2856135: OPC protrusion_1; Rattus norvegicus; RNA-Seq
library_construction_protocol: Total RNA from OPC soma and membrane protrusions was isolated using the Quick-RNA MicroPrep kit (Zymo Research) according with manufacturer's instructions.  The OPC soma fraction was isolated by scrapping the upper side of the insert with a cotton swab.  Total RNA was further isolated from the cotton swab by dipping it in ZR RNA lysis buffer.  RNA from membrane protrusions was collected from the bottom side of the insert, which was pushed down into a well containing lysis buffer.  RNA from different experiments was pooled and concentrated using the RNA Clean & Concentrator-5 kit (Zymo Research). 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 5 μg 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® Ultra™ Directional RNA Library Prep Kit for Illumina® (E7420L) 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 1.0: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', Index primer: 5'-CAAGCAGAAGACGGCATACGAGAT(INDEX)CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'. Indices were according to the eight bases tags developed by WTCHG (Lamble et al., 2013). 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.