GSM9743455: FUS_R521L_ACMS_Rep3; Homo sapiens; RNA-Seq
Total RNA from entire MFC MN cultures was extracted and purified at 48 DIV as illustrated in associated manuscript: Fig. 1J either after ACMS (at 25 Hz, perpendicular field orientation and 10 mT) or from untreated controls (UT). The preparation was performed with the Qiagen RNeasy® Plus kit (Cat no. 74134) according to the manufacture's manual and typically resulting in 5-10 µg of total RNA dissolved in a total volume of 80 µl of DNAse/RNAse-free water. Concentration and integrity of each RNA sample was analyzed on an Agilent® 2100 Bioanalyzer (assay class: eukaryote total RNA nano, version 2.6), all resultant RNA integrity numbers (RIN) were in the range of 8.7 - 9.7, indicating high purity and integrity of all samples. Messenger RNA was isolated from 500 ng of each total RNA sample (see above) by poly-dT enrichment using the New England Biolabs® (NEB) Next Poly-A mRNA Magnetic Isolation Module according to the manufacturer's instructions. Samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB). For ligation, Next Adapters for Illumina of the NEB Next Multiplex Oligos for Illumina kit were used. After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 0.9:1 to the samples. Unique dual indexing was performed during the following PCR enrichment (12 cycles) using amplification primers carrying the same sequence for i7 and i5 index (i5: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, i7: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (0.9:1), libraries were quantified using the Fragment Analyzer (Agilent®).