NextSeq 2000 sequencing; RNA seq of HEK293 treated with Blebbistatin or expressing C9orf72 associated Di Peptide Repeats (DPRs)
RNA seq of HEK293 treated with Blebbistatin or expressing C9orf72 associated Di Peptide Repeats (DPRs)
Samples were lysed on plate using RNeasy kit (Qiagen) Lysis buffer. RNA was extracted using RNeasy kit (Qiagen) using manufacturer protocol. DNA was removed in column treatment and in-solution with DNAse I. Individual libraries were generated from 500ng of each total RNA sample using the QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) for Illumina (Lexogen Inc, #015). Libraries were prepared according to the QuantSeq 3' mRNA-seq Library Prep Kit (FWD) protocol. The QuantSeq protocol generates only one fragment per transcript, resulting in extremely accurate gene expression values, and the sequences obtained are close to the 3' end of the transcripts. Library generation was initiated by oligo-dT priming. The primer already contains Illumina-compatible linker sequences. After first strand synthesis, the RNA was removed and second strand synthesis initiated by random priming and a DNA polymerase. With the QuantSeq FWD kit the Read 1 linker sequence is contained in the second strand synthesis primer so NGS reads are generated towards the poly (A) tail and directly correspond to the mRNA sequence. Second strand synthesis was followed by a magnetic bead-based purification step before cDNA libraries were amplified for 13 cycles introducing the sequences required for cluster generation and index sequences to allow multiplexing of sequencing libraries. After amplification a final bead-purification removed any excess primers or adapterdimers that may interfere with sequencing.