E-MTAB-4732 - 3'-end targeted sequencing of 6 control and 6 TDP-43 KD samples in HEK293
Last updated on 21 April 2017, released on 1 May 2017
HEK293 FlpIn T-Rex cells were maintained in Dulbeccos Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), supplemented with 3 g/ml blasticidine and 50 g/ml zeocin. For the siRNA-induced knockdown of TDP-43, 20 nM of TDP-43 stealth siRNA was mixed with 10 L of RNAiMAX following the manufacturers reverse transfection protocol and added to a 10 cm dish of HEK FlpIn cells. For the non-targeting siRNA also 20 nM was used, to distinguish off-target effects from biologically relevant ones. After the first 24 hrs of transfection, the medium was replaced with DMEM with 10% FBS and after an additional 24 hrs, the cells were collected for analysis. RNA was extracted using the Direct-zol RNA kit and an in-column DNase digestion step was performed at room temperature for 15 minutes. The poly(A)seq libraries for samples, that were transfected with either non-targeting siRNA or TARDBP siRNA, were generated using the reverse QuantSeq 3mRNA-Seq kit. Libraries were prepared from 500 ng of total RNA. In the protocol one fragment per transcript was generated, which resulted in extremely accurate gene expression values. For the initial step of this kit, oligodT priming, including Illumina-compatible linker sequences, was carried out. The second strand synthesis was followed by purification with magnetic beads. Barcodes were introduced during the PCR amplification step as standard external barcodes. Single-end sequencing (60 nts) was performed on a Illumina GA-2 with a Rapid Run flow-cell. This kit makes use of a custom sequencing primer that anneals to a linker sequence previously introduced in the oligo(dT) priming step for reverse transcription.
RNA-seq of coding RNA, case control design
High-resolution RNA maps suggest common principles of splicing and polyadenylation regulation by TDP-43. Gregor Rot, Zhen Wang, Ina Huppertz, Miha Modic, Tina Lence, Martina Hallegger, Nejc Haberman, Tomaz Curk, Christian von Mering, Jernej Ule.