Illumina MiSeq sequencing; modified iCLIP experiment with the addition of a 5'marker to identify read-through reads
modified iCLIP experiment with the addition of a 5'marker to identify read-through reads
The modified iCLIP protocol is based on the previously described protocol with modifications that enable the definition of read–through cDNAs (Huppertz el al., 2014). HEK293 cells were crosslinked with 0.15 mJ/cm2 254 nm UV light. To prepare the cell lysate, the cells were lysed with 1 ml iCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, final concentration 2mg/ml) and sonicated (Bioruptor, 5x5 sec on/off).  For the iCLIP library preparation of PTBP1, the sonicated lysates were initially centrifuged and the supernatant was kept as the first lysate. The remaining cell pellet was dissolved in 50 µL MSB lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaH2PO4, 7 M urea, 1 mM DTT). After the pellet had dissolved, the mixture was diluted with H2O to 1000 μL and an additional centrifugation was performed (second lysate). The two lysates were combined and incubated with RNase I (AM2295, Thermo Scientific) at 37°C for 3 min at 4 U/ml. Protein A Dynabeads were used for the IP of PTBP1. For each IP, 100 µl of beads were washed in iCLIP lysis buffer and incubated with the mouse monoclonal BB7 serum anti-PTBP1 (a gift from C. Smith, available from ATCC CRL-2501). The samples were then rotated at 4°C for 2 h (final urea concentration 175 mM).  The beads were washed with high-salt washing buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). After the first round of washes, the samples proceed through 3´ adapter addition, an additional phosphorylation (0.2 µl PNK, 0.4 µl cold ATP (1 mM), 0.4 µl 10x PNK buffer, 3 µl water) and a 5´marker ligation (6 µl water, 5 µl 4X ligation buffer, 2 µl RNA ligase, 1 µl RNasin, 2 µl 5´ marker (100 µM), 4 µl PEG400). The sequence of the 5´ marker is CAGUCCGACGAUC, which corresponds to the Illumina short RNA 5´ Adapter (RA5), part #15013205; this sequence is not complementary to the primers used for amplification of iCLIP cDNA libraries (Huppertz el al., 2014).  We followed the protocol as described in Huppertz et al., 2014
Experimental Factor: immunoprecipitate
anti-PTBP1