title: Illumina HiSeq 4000 sequencing; CLIP-seq to identify transcriptome-wide TDP-43-RNA binding sites in 293HEK FlipIn cells
design: CLIP-seq to identify transcriptome-wide TDP-43-RNA binding sites in 293HEK FlipIn cells
library_construction_protocol: This experiment identified TDP-43-RNA binding sites in 293 Hek FlipIn cells. Cells were grown to 80% confluence, transgene was induced by doxycycline for 24h , UV crosslinked on ice and then lysed in RIPA buffer.   Hexanediol treated samples were pretreated with 8% 1,6- and 2,5-Hexanediol for 5 min at 37 degrees and then cross-linked on ice.  0.4 Units of RNaseI and 4 Units Turbo DNase were added per 1 ml of cell lysate at 1mg/ml protein concentration for RNA fragmentation. Negative controls (no-UV) were prepared. Antibodies against GFP coupled to magnetic Protein G beads or  GFP-TRAP_MA beads (Chromotek, gtma-20) were used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labelled IRL3 adaptor. For some experiments we chose to multiplex a maximum of 3 samples before the protein gel which required for the ligation step a bar-coded unlabelled L3 adaptor. The complexes were then size-separated by SDS-PAGE, blotted onto nitrocellulose and visualised by Odyssey scanning. For the multiplexed sample 1 replicated was run in parallel with the IRL3 to allow quality control of the RNP complex on the membrane and to help with cutting of the bands. RNA was released from membrane by proteinase K digestion and recovered by precipitation. cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and AMPure XP beads purification (Beckman- Coulter, USA), then circularised using Circligase II (Epicentre) followed by AMPure XP beads purification. After PCR amplification, libraries were size-selected with Ampure beads (if necessary by gel-purification) and quality controlled for sequencing. For the 'RNase experiment' we use 4 units Turbo DNase and  0.1 (low), 0.4 (medium) or 2 units (high) per 1 ml of lysate at 1mg/ml protein concentration. cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and AMPure XP beads purification (Beckman- Coulter, USA), then circularised using Circligase II (Epicentre) followed by AMPure XP beads purification. After PCR amplification, libraries were size-selected with Ampure beads (if necessary by gel-purification) and quality controlled for sequencing.