GSM2888354: pNSC_3D_ab42+IL4_R3; Homo sapiens; RNA-Seq
For cells from 3D gels, we used Norgen Total RNA Isolation Kit. RNA isolation performed according to the protocol suggest by the company except; 3 gels lysed in 1ml RL buffer with 10 µL beta-mercaptoethanol. Centrifuged at 10000g for 5 min at RT and supernatant (~930 µL) transfered to a new eppendorf. 580 µL %96-99 ethanol added and mixed throughly. After passing through samples on columns, washings and DNase I treatment done accroding to the protocol suggests and RNA eluted in50 µL  nuclease free water. Samples ran on Agilent Bioanalyser for RNA quality, samples with RNA integrity >= 9.0 used for library preparation. Total RNA with an integrity number of ≥ 8 was used. mRNA was isolated from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module. After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB)
GEO Accession
GSM2888354